IL1RL-1 as a Cardiovascular Disease Marker and Therapeutic Target

ABSTRACT

This invention pertains to methods and compositions for the diagnosis and treatment of cardiovascular conditions. More specifically, the invention relates to isolated molecules that can be used to diagnose and/or treat cardiovascular conditions including cardiac hypertrophy, myocardial infarction, stroke, arteriosclerosis, and heart failure.

RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 13/788,276, filed on Mar. 7, 2013 (issued as U.S.Pat. No. 8,748,116), which is a continuation application of U.S. patentapplication Ser. No. 13/150,749, filed on Jun. 1, 2011 (issued as U.S.Pat. No. 8,597,958), which is a continuation application of U.S. patentapplication Ser. No. 12/614,970, filed on Nov. 9, 2009 (issued as U.S.Pat. No. 7,989,210), which is a continuation application of U.S. patentapplication Ser. No. 10/435,482, filed on May 9, 2003 (issued as U.S.Pat. No. 7,670,769), which claims the benefit under 35 U.S.C. § 119 ofU.S. Provisional Patent Application Ser. No. 60/379,173, filed May 9,2002, the contents of each of which are incorporated herein by referencein their entirety.

GOVERNMENT SUPPORT

The work resulting in this invention was supported in part by NIH GrantNos. HL69484, HL63927, and HL052320. Accordingly, the U.S. Governmentmay be entitled to certain rights in the invention.

FIELD OF THE INVENTION

This invention relates to methods and compositions for the diagnosis andtreatment of cardiovascular conditions. More specifically, the inventionrelates to isolated molecules that can be used to treat cardiovascularconditions including cardiac hypertrophy, myocardial infarction, stroke,arteriosclerosis, and heart failure.

BACKGROUND OF THE INVENTION

Despite significant advances in therapy, cardiovascular disease remainsthe single most common cause of morbidity and mortality in the developedworld. Thus, prevention and therapy of cardiovascular conditions such asmyocardial infarction and stroke is an area of major public healthimportance. Currently, several risk factors for future cardiovasculardisorders have been described and are in wide clinical use in thedetection of subjects at high risk. Such screening tests includeevaluations of total and HDL cholesterol levels. However, a large numberof cardiovascular disorders occur in subjects with apparently low tomoderate risk profiles, and ability to identify such patients islimited. Moreover, accumulating data suggests that the beneficialeffects of certain preventive and therapeutic treatments for patients atrisk for or known to have cardiovascular disorders differs in magnitudeamong different patient groups. At this time, however, data describingdiagnostic tests to determine whether certain therapies can be expectedto be more or less effective are lacking.

SUMMARY OF THE INVENTION

This invention provides methods and compositions for the diagnosis andtreatment of cardiovascular conditions. More specifically, a gene wasidentified that is upregulated in cardiac cells when the cells aresubjected to mechanically-induced deformation. In view of thesediscoveries, it is believed that the molecules of the present inventioncan be used to treat cardiovascular (including vascular) conditions,including cardiac hypertrophy, myocardial infarction, stroke,arteriosclerosis, and heart failure.

Additionally, methods for using these molecules in the diagnosis of anyof the foregoing cardiovascular (including vascular) conditions, arealso provided.

Furthermore, compositions useful in the preparation of therapeuticpreparations for the treatment of the foregoing conditions, are alsoprovided.

The present invention thus involves, in several aspects, polypeptides,isolated nucleic acids encoding those polypeptides, functionalmodifications and variants of the foregoing, useful fragments of theforegoing, as well as therapeutics and diagnostics relating thereto.

According to one aspect of the invention, a method of diagnosing acondition characterized by aberrant expression of a nucleic acidmolecule or an expression product thereof (or of unique fragments of theforegoing molecules thereof), is provided. The method involvescontacting a biological sample from a subject with an agent, whereinsaid agent specifically binds to said nucleic acid molecule, anexpression product thereof, or a fragment of an expression productthereof, and measuring the amount of bound agent and determiningtherefrom if the expression of said nucleic acid molecule or of anexpression product thereof is aberrant, aberrant expression beingdiagnostic of the disorder, wherein the nucleic acid molecule isInterleukin 1 Receptor-Like 1 (IL1RL-1, also known as T1/ST2, ST2, andFit-1, SEQ ID NOs: 1 and 2 for the soluble form and SEQ ID NOs: 3 and 4for the membrane form). The terms IL1RL-1, T1/ST2, ST2, and Fit-1, areused interchangeably hereinafter throughout the specification. In someembodiments, the disorder is a cardiovascular condition selected fromthe group consisting of myocardial infarction, stroke, arteriosclerosis,and heart failure. In one embodiment, the disorder is cardiachypertrophy. In another embodiment, the disorder is heart failure. Incertain embodiments, biological samples include biopsy samples, andbiological fluids such as blood/serum.

According to still another aspect of the invention, a method fordetermining a stage (e.g, regression, progression or onset) of acardiovascular condition in a subject characterized by aberrantexpression of a nucleic acid molecule or an expression product thereof(or of unique fragments of the foregoing molecules thereof), isprovided. The method involves monitoring a sample from a patient for aparameter selected from the group consisting of (i) a IL1RL-1 nucleicacid molecule (or a unique fragment thereof), (ii) a polypeptide encodedby the IL1RL-1 nucleic acid, (iii) a peptide derived from thepolypeptide (or of a unique fragment thereof), and (iv) an antibodywhich selectively binds the polypeptide or peptide (or a unique fragmentthereof), as a determination of a stage (e.g., regression, progressionor onset) of said cardiovascular condition in the subject. In someembodiments, the sample is a biological fluid or a tissue as describedin any of the foregoing embodiments. In certain embodiments, the step ofmonitoring comprises contacting the sample with a detectable agentselected from the group consisting of (a) an isolated nucleic acidmolecule which selectively hybridizes under stringent conditions to thenucleic acid molecule of (i), (b) an antibody which selectively bindsthe polypeptide of (ii), or the peptide of (iii), and (c) a polypeptideor peptide which selectively binds the antibody of (iv). The antibody,polypeptide, peptide, or nucleic acid can be labeled with a detectablelabel such as a radioactive label or an enzyme. In further embodiments,the method comprises monitoring (assaying) the sample for the peptide.In still further embodiments, monitoring the sample occurs over a periodof time.

According to another aspect of the invention, a kit is provided. The kitcomprises a package containing an agent that selectively binds to any ofthe foregoing IL1RL-1 isolated nucleic acids, or expression productsthereof, and a control for comparing to a measured value of binding ofsaid agent any of the foregoing isolated nucleic acids or expressionproducts thereof. In some embodiments, the control is a predeterminedvalue for comparing to the measured value. In certain embodiments, thecontrol comprises an epitope of the expression product of any of theforegoing isolated nucleic acids.

According to one aspect of the invention, a method for treating acardiovascular condition is provided. The method involves administeringto a subject in need of such treatment a IL1RL-1 molecule, in an amounteffective to treat the cardiovascular condition. In certain embodiments,the cardiovascular condition is selected from the group consisting ofmyocardial infarction, stroke, arteriosclerosis, and heart failure. Insome embodiments, the method further comprises co-administering an agentselected from the group consisting of an anti-inflammatory agent, ananti-thrombotic agent, an anti-platelet agent, a fibrinolytic agent, alipid reducing agent, a direct thrombin inhibitor, a glycoproteinIIb/IIIa receptor inhibitor, an agent that binds to cellular adhesionmolecules and inhibits the ability of white blood cells to attach tosuch molecules, a calcium channel blocker, a beta-adrenergic receptorblocker, a cyclooxygenase-2 inhibitor, or an angiotensin systeminhibitor.

According to another aspect of the invention, a method for treatingcardiac hypertrophy is provided. The method involves administering to asubject in need of such treatment an agent that increases expression ofa IL1RL-1 nucleic acid molecule, or an expression product thereof, in anamount effective to treat cardiac hypertrophy in the subject.

According to a further aspect of the invention, a method for treating asubject to reduce the risk of a cardiovascular condition developing inthe subject is provided. The method involves administering to a subjectthat expresses aberrant levels of a IL1RL-1 molecule, an agent forreducing the risk of the cardiovascular disorder in an amount effectiveto lower the risk of the subject developing a future cardiovasculardisorder, wherein the agent is an anti-inflammatory agent, ananti-thrombotic agent, an anti-platelet agent, a fibrinolytic agent, alipid reducing agent, a direct thrombin inhibitor, a glycoproteinIIb/IIIa receptor inhibitor, an agent that binds to cellular adhesionmolecules and inhibits the ability of white blood cells to attach tosuch molecules, a calcium channel blocker, a beta-adrenergic receptorblocker, a cyclooxygenase-2 inhibitor, or an angiotensin systeminhibitor. In certain embodiments, the subject is otherwise free ofsymptoms calling for treatment with the agent.

According to one aspect of the invention, a method for identifying acandidate agent useful in the treatment of a cardiovascular condition isprovided. The method involves determining expression of IL1RL-1 moleculein a cardiac cell or tissue under conditions which, in the absence of acandidate agent, permit a first amount of expression of the IL1RL-1molecule, contacting the cardiac cell or tissue with the candidateagent, and detecting a test amount of expression of the IL1RL-1molecule, wherein a decrease in the test amount of expression in thepresence of the candidate agent relative to the first amount ofexpression indicates that the candidate agent is useful in the treatmentof the cardiovascular condition. In important embodiments the IL1RL-1molecule is any molecule of SEQ ID NO.:1-4. In certain embodiments, thecardiovascular condition is selected from the group consisting ofcardiac hypertrophy (e.g., maladaptive hypertrophy), myocardialinfarction, stroke, arteriosclerosis, and heart failure.

According to another aspect of the invention, a pharmaceuticalcomposition is provided. The composition comprises an agent comprisingan IL1RL-1 isolated nucleic acid molecule (SEQ ID NO.:1 or 3), or anexpression product thereof (e.g., SEQ ID NO.:2 or 4), in apharmaceutically effective amount to treat a cardiovascular condition,and a pharmaceutically acceptable carrier. In certain embodiments, thecardiovascular condition is selected from the group consisting ofcardiac hypertrophy, myocardial infarction, stroke, arteriosclerosis,and heart failure.

According to a further aspect of the invention, methods for preparingmedicaments useful in the treatment of a cardiovascular condition arealso provided. The medicaments preferably contain an effective amount ofat least one of the foregoing molecules or compositions.

According to still another aspect of the invention, a solid-phasenucleic acid molecule array is provided. The array consists essentiallyof a set of nucleic acid molecules, expression products thereof, orfragments (of either the nucleic acid or the polypeptide molecule)thereof, wherein at least a IL1RL-1 molecule (including expressionproducts thereof, or fragments thereof), are fixed to a solid substrate.In some embodiments, the solid-phase array further comprises at leastone control nucleic acid molecule.

In certain embodiments, the solid substrate includes a material selectedfrom the group consisting of glass, silica, aluminosilicates,borosilicates, metal oxides such as alumina and nickel oxide, variousclays, nitrocellulose, and nylon. Preferably the substrate is glass. Insome embodiments, the nucleic acid molecules are fixed to the solidsubstrate by covalent bonding.

According to another aspect of the invention, a method for evaluatingthe likelihood that a subject will benefit from treatment with an agentfor reducing the risk of a cardiovascular condition, is provided. Inimportant embodiments the agent is selected from the group consisting ofan anti-inflammatory agent, an antithrombotic agent, an anti-plateletagent, a fibrinolytic agent, a lipid reducing agent, a direct thrombininhibitor, a glycoprotein IIb/IIIa receptor inhibitor, an agent thatbinds to cellular adhesion molecules and inhibits the ability of whiteblood cells to attach to such molecules, a calcium channel blocker, abeta-adrenergic receptor blocker, a cyclooxygenase-2 inhibitor, and anangiotensin system inhibitor. The method involves obtaining a level of aIL1RL-1 molecule in the subject, and comparing the level of the IL1RL-1molecule to a predetermined value specific for the diagnosis of acardiovascular condition. The level of the IL1RL-1 molecule incomparison to the predetermined value is indicative of whether thesubject will benefit from treatment with said agent. In certainembodiments, the predetermined value specific for the diagnosis of acardiovascular condition is a plurality of predetermined marker levelranges and said comparing step comprises determining in which of saidpredetermined marker level ranges said subjects level falls. Thecardiovascular condition can be a condition selected from the groupconsisting of cardiac hypertrophy, myocardial infarction, stroke,arteriosclerosis, and heart failure.

In another aspect of the invention a method for predicting outcome of acardiovascular condition is provided. The method involves obtaining alevel of a IL1RL-1 molecule in the subject, and comparing the level ofthe IL1RL-1 molecule to a predetermined value specific for the predictedoutcome of a cardiovascular condition. The level of the IL1RL-1 moleculein comparison to the predetermined value is indicative of whether thesubject will have a good/positive outcome or will have a bad/negativeoutcome. In some embodiments a high level of the IL1RL-1 molecule mightindicate a negative outcome while a low level might indicate a positiveoutcome. In certain embodiments, the predetermined value specific forthe predicted outcome of a cardiovascular condition is a plurality ofpredetermined marker level ranges and said comparing step comprisesdetermining in which of said predetermined marker level ranges saidsubjects level falls. The cardiovascular condition can be a conditionselected from the group consisting of cardiac hypertrophy, myocardialinfarction, stroke, arteriosclerosis, and heart failure.

Any sequence of an IL1RL-1 molecule may be used in any of the aspectsand embodiments of the invention. For instance, this includes thenucleotide sequences set forth as SEQ ID NOs.: 5 and 7, in addition tothe nucleotide sequences set forth as SEQ ID NOs.: 1 and 3. This furtherincludes the predicted amino acid sequences set forth as SEQ ID NOs.: 6and 8, in addition to the predicted amino acid sequences set forth asSEQ ID NOs.: 2 and 4.

These and other objects of the invention will be described in furtherdetail in connection with the detailed description of the invention.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the nucleotide sequence of the human IL1RL1 (Soluble)cDNA.

SEQ ID NO:2 is the predicted amino acid sequence of the translationproduct of the human IL1RL1 (Soluble) cDNA (SEQ ID NO:1).

SEQ ID NO:3 is the nucleotide sequence of the human IL1RL1 (Membrane)cDNA.

SEQ ID NO:4 is the predicted amino acid sequence of the translationproduct of the human IL1RL1 (Membrane) (SEQ ID NO:3).

SEQ ID NO:5 is the nucleotide sequence of the rat Fit-1S cDNA.

SEQ ID NO:6 is the predicted amino acid sequence of the translationproduct of rat Fit-1S cDNA (SEQ ID NO:5).

SEQ ID NO:7 is the nucleotide sequence of the rat Fit-1M cDNA.

SEQ ID NO:8 is the predicted amino acid sequence of the translationproduct of the rat Fit-1M cDNA (SEQ ID NO:7).

DESCRIPTION OF DRAWINGS

FIG. 1 depicts by a Northern Blot the effects of 8% cyclic mechanicalstrain on the expression of Fit-1 in cultured cardiac myocytes over thecourse of time.

FIG. 2 depicts by a Northern Blot the effects of 8% cyclic mechanicalstrain, angiotensin receptor blockade, angiotensin II, IL-1b, andphorbol ester, on the expression of IL1RL-1 in cultured cardiac myocytesover the course of time.

FIG. 3 depicts by a Northern Blot the effects of 8% cyclic mechanicalstrain, hydrogen peroxide, and TIRON, on the expression of IL1RL-1 incultured cardiac myocytes over the course of time.

FIG. 4 depicts by a Northern Blot the effects of actinomycin D andcyclohexamide on the induction of IL1RL-1 expression during an 8% cyclicmechanical strain on cardiac myocytes over the course of time.

FIG. 5 depicts by a Northern Blot the effects of 8% cyclic mechanicalstrain alone and in combination with IL-1b, and phorbol ester in theabsence of strain, on the expression of IL1RL-1 in cultured cardiacmyocytes over the course of time.

FIG. 6 depicts by a Northern Blot the effects of an 8% cyclic mechanicalstrain on the expression of vacuolar ATPase in cultured cardiac myocytesover the course of time.

FIG. 7 depicts a kit embodying features of the present invention.

FIG. 8 depicts early (left) and late (right) time course of the mRNAinduction of T2/ST2 by mechanical strain in cardiac myocytes. Maximalinduction occurs at 3 hours, is sustained for 9 hours and declines by 15hours. Top panels, T1/ST2 RNA; bottom panels, ethidium bromide. No str,no strain.

FIG. 9 depicts mRNA induction of T1/ST2 by mechanical strain (8%),interleukin-1 (10 ng/ml) and phorbol ester (PMA, 200 nM) at 1 and 3hours. PMA>strain>IL-1. Top panel, T1/ST2 mRNA, bottom panel, ethidiumbromide.

FIG. 10 shows that T1/ST2 may be a gene induced by NF-κB activationduring IL-1/IL-receptor signaling in cardiac myocytes. IL-1 and straininduced T1/ST2 mRNA in the presence of infection with control adenovirus(left). With infection of IκB adenovirus (right), which decreases NF-κBDNA binding activity, the IL-1 induction of T1/ST2 was blocked. Thestrain induction of T1/ST2 was partially blocked by IκB adenovirusinfection suggesting another pathway for induction of T1/ST2 by strain.Top panel, T1/ST2 mRNA; bottom panel, ethidium bromide.

FIG. 11 shows expression of T1/ST2 protein following myocardialinfarction in mice by immunohistochemistry at 1 day but not 3 days afterinfarction. 40× magnification.

FIG. 12A shows in graphical form ST2 protein levels in the systemiccirculation of human patients post myocardial infarction on day 1, day14, and day 90 post myocardial infarction. ST2 protein was significantlyincreased on day 1 post myocardial infarction compared to day 14 and day90.

FIG. 12B is a graph of a Linear regression analysis demonstrating asignificant positive relationship (p<0.001) between circulating ST2protein and creatine kinase 1 day post myocardial infarction. LogST2=0.454(log CK)−1.07.

FIG. 12C is a graph of the quartile analysis of circulating ST2 proteinlevels day 1 post myocardial infarction and ejection fraction. Lowejection fraction is associated with high ST2 protein levels.

FIG. 12D is a graph of a Linear regression analysis showing therelationship between circulating ST2 protein levels day 1 postmyocardial infarction and ejection function.

FIG. 13 shows that elevated baseline levels of ST2 were indicative ofhigher mortality through 30 days of follow-up (log-rank, p=0.0009).

DETAILED DESCRIPTION

The invention involves the discovery of a number of genes that areupregulated in cardiac cells when the cells are subjected to amechanically-induced strain deformation. In view of this discovery, itis believed that the molecules of the present invention can be used totreat cardiovascular conditions including cardiac hypertrophy,myocardial infarction, stroke, arteriosclerosis, and/or heart failure.

Additionally, methods for using these molecules in the diagnosis of anyof the foregoing cardiovascular conditions, are also provided.

Furthermore, compositions useful in the preparation of therapeuticpreparations for the treatment of the foregoing conditions, are alsoprovided.

“Upregulated,” as used herein, refers to increased expression of a geneand/or its encoded polypeptide. “Increased expression” refers toincreasing (i.e., to a detectable extent) replication, transcription,and/or translation of any of the nucleic acids of the invention(IL1RL-1, SEQ ID NOs.:1, 3), since upregulation of any of theseprocesses results in concentration/amount increase of the polypeptideencoded by the gene (nucleic acid). Conversely, “downregulation,” or“decreased expression” as used herein, refers to decreased expression ofa gene and/or its encoded polypeptide. The upregulation ordownregulation of gene expression can be directly determined bydetecting an increase or decrease, respectively, in the level of mRNAfor the gene, or the level of protein expression of the gene-encodedpolypeptide, using any suitable means known to the art, such as nucleicacid hybridization or antibody detection methods, respectively, and incomparison to controls.

A “cardiac cell”, as used herein, refers to a cardiomyocyte.

A “molecule,” as used herein, embraces both “nucleic acids” and“polypeptides.”

“Expression,” as used herein, refers to nucleic acid and/or polypeptideexpression.

As used herein, a “subject” is a mammal or a non-human mammal. In allembodiments human nucleic acids, polypeptides, and human subjects arepreferred. It is believed that the results obtained using the human andrat molecules described elsewhere herein are predictive of the resultsthat may be obtained using other homologous sequences.

In general, homologs and alleles typically will share at least 80%nucleotide identity and/or at least 85% amino acid identity to thecharacterized human sequences of the invention. In further instances,homologs and alleles typically will share at least 90%, 95%, or even 99%nucleotide identity and/or at least 95%, 98%, or even 99% amino acididentity to the characterized human sequences, respectively. Thehomology can be calculated using various, publicly available softwaretools developed by NCBI (Bethesda, Md.). Exemplary tools include theheuristic algorithm of Altschul S F, et al., (J Mol Biol, 1990,215:403-410), also known as BLAST. Pairwise and ClustalW alignments(BLOSUM30 matrix setting) as well as Kyte-Doolittle hydropathic analysiscan be obtained using public (EMBL, Heidelberg, Germany) and commercial(e.g., the MacVector sequence analysis software from Oxford MolecularGroup/Genetics Computer Group, Madison, Wis., Accelrys, Inc., San Diego,Calif.). Watson-Crick complements of the foregoing nucleic acids alsoare embraced by the invention.

In screening for related genes, such as homologs and alleles of thesequences described elsewhere herein, a Southern blot may be performedusing stringent conditions, together with a probe. The term “stringentconditions,” as used herein, refers to parameters with which the art isfamiliar. With nucleic acids, hybridization conditions are said to bestringent typically under conditions of low ionic strength and atemperature just below the melting temperature (Tm) of the DNA hybridcomplex (typically, about 3° C. below the T. of the hybrid). Higherstringency makes for a more specific correlation between the probesequence and the target. Stringent conditions used in the hybridizationof nucleic acids are well known in the art and may be found inreferences which compile such methods, e.g. Molecular Cloning: ALaboratory Manual, J. Sambrook, et al., eds., Second Edition, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, orCurrent Protocols in Molecular Biology, F. M. Ausubel, et al., eds.,John Wiley & Sons, Inc., New York. An example of “high stringencyconditions” is hybridization at 65° C. in 6×SSC. Another example of highstringency conditions is hybridization at 65° C. in hybridization bufferthat consists of 3.5×SSC, 0.02% Ficoll, 0.02% polyvinyl pyrolidone,0.02% Bovine Serum Albumin, 2.5 mM NaH₂PO₄[pH7], 0.5% SDS, 2 mM EDTA.(SSC is 0.015M sodium chloride/0.15M sodium citrate, pH7; SDS is sodiumdodecyl sulphate; and EDTA is ethylenediaminetetraacetic acid). Afterhybridization, the membrane upon which the DNA is transferred is washedat 2×SSC at room temperature and then at 0.1×SSC/0.1×SDS at temperaturesup to 68° C. In a further example, an alternative to the use of anaqueous hybridization solution is the use of a formamide hybridizationsolution. Stringent hybridization conditions can thus be achieved using,for example, a 50% formamide solution and 42° C. There are otherconditions, reagents, and so forth which can be used, and would resultin a similar degree of stringency. The skilled artisan will be familiarwith such conditions, and thus they are not given here. It will beunderstood, however, that the skilled artisan will be able to manipulatethe conditions in a manner to permit the clear identification ofhomologs and alleles of IL1RL-1 nucleic acids of the invention. Theskilled artisan also is familiar with the methodology for screeningcells and libraries for expression of such molecules which then areroutinely isolated, followed by isolation of the pertinent nucleic acidmolecule and sequencing.

Given the teachings herein of full-length human and rat cDNA clones,other mammalian sequences such as (mouse, bovine, etc.) cDNAscorresponding to the related human and rat nucleic acids can be isolatedfrom cDNA libraries using standard colony hybridization techniques, orcan be identified using a homology search, for example, in GenBank usingany of the algorithms described elsewhere herein or known in the art.For example, sequences with GenBank Accession numbers Y07519.1 andD13695.1 for the mouse IL1RL-1 homologs, can be used interchangeablywith the homologous rat sequences of the invention, in all aspects ofthe invention without departing from the essence of the invention.

As used herein with respect to nucleic acids, the term “isolated” means:(i) amplified in vitro by, for example, polymerase chain reaction (PCR);(ii) recombinantly produced by cloning; (iii) purified, as by cleavageand gel separation; or (iv) synthesized by, for example, chemicalsynthesis. An isolated nucleic acid is one which is readily manipulatedby recombinant DNA techniques well known in the art. Thus, a nucleotidesequence contained in a vector in which 5′ and 3′ restriction sites areknown or for which polymerase chain reaction (PCR) primer sequences havebeen disclosed is considered isolated, but a nucleic acid sequenceexisting in its native state in its natural host is not. An isolatednucleic acid may be substantially purified, but need not be. Forexample, a nucleic acid that is isolated within a cloning or expressionvector is not pure in that it may comprise only a tiny percentage of thematerial in the cell in which it resides. Such a nucleic acid isisolated, however, as the term is used herein because it is readilymanipulated by standard techniques known to those of ordinary skill inthe art.

According to the invention, expression of any of the foregoing IL1RL-1nucleic acids of the present invention, including unique fragments ofthe foregoing, can be determined using different methodologies. A“unique fragment,” as used herein, with respect to a nucleic acid is onethat is a “signature” for the larger nucleic acid. For example, theunique fragment is long enough to assure that its precise sequence isnot found in molecules within the human genome outside of the sequencefor each nucleic acid defined above. Those of ordinary skill in the artmay apply no more than routine procedures to determine if a fragment isunique within the human genome. Unique fragments, however, excludefragments completely composed of nucleotide sequences previouslypublished as of the filing date of this application.

Unique fragments can be used as probes in Southern and Northern blotassays to identify such nucleic acids, or can be used in amplificationassays such as those employing PCR. As known to those skilled in theart, large probes such as 200, 250, 300 or more nucleotides arepreferred for certain uses such as Southern and Northern blots, whilesmaller fragments will be preferred for other uses such as PCR. Uniquefragments also can be used to produce fusion proteins for generatingantibodies, or determining binding of the polypeptide fragments, or forgenerating immunoassay components. Likewise, unique fragments can beemployed to produce nonfused fragments of, for example, the IL1RL-1polypeptides, useful, for example, in the preparation of antibodies,immunoassays or therapeutic applications. Unique fragments further canbe used as antisense molecules to inhibit the expression of theforegoing nucleic acids and polypeptides respectively.

As will be recognized by those skilled in the art, the size of theunique fragment will depend upon its conservancy in the genetic code.Thus, some regions of SEQ ID NOs: 1, and 3, and complements will requirelonger segments to be unique while others will require only shortsegments, typically between 12 and 32 nucleotides long (e.g., 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31and 32 bases) or more, up to the entire length of each of the disclosedsequences. As mentioned above, this disclosure intends to embrace eachand every fragment of each sequence, beginning at the first nucleotide,the second nucleotide and so on, up to 8 nucleotides short of the end,and ending anywhere from nucleotide number 8, 9, 10 and so on for eachsequence, up to the very last nucleotide, (provided the sequence isunique as described above). For example, virtually any segment of theregion of SEQ ID NO:1 beginning at nucleotide 1 and ending at nucleotide1357, or SEQ ID NO:3 beginning at nucleotide 1 and ending at nucleotide2058, or complements thereof, that is 20 or more nucleotides in lengthwill be unique. Those skilled in the art are well versed in methods forselecting such sequences, typically on the basis of the ability of theunique fragment to selectively distinguish the sequence of interest fromother sequences in the human genome of the fragment to those on knowndatabases typically is all that is necessary, although in vitroconfirmatory hybridization and sequencing analysis may be performed.

In certain aspects, the invention embraces antisense oligonucleotidesthat selectively bind to a nucleic acid molecule encoding a polypeptide,to decrease the polypeptide's activity.

As used herein, the terms “antisense molecules,” “antisenseoligonucleotide,” and “antisense” describe an oligonucleotide that is anoligoribonucleotide, oligodeoxyribonucleotide, modifiedoligoribonucleotide, or modified oligodeoxyribonucleotide whichhybridizes under physiological conditions to DNA comprising a particulargene or to an mRNA transcript of that gene and, thereby, inhibits thetranscription of that gene and/or the translation of that mRNA. Theantisense molecules are designed so as to interfere with transcriptionor translation of a target gene upon hybridization with the target geneor transcript. Those skilled in the art will recognize that the exactlength of an antisense oligonucleotide and its degree of complementaritywith its target will depend upon the specific target selected, includingthe sequence of the target and the particular bases which comprise thatsequence. It is preferred that an antisense oligonucleotide beconstructed and arranged so as to bind selectively with a target underphysiological conditions, i.e., to hybridize substantially more to thetarget sequence than to any other sequence in the target cell underphysiological conditions. Based upon SEQ ID NOs: 1, and 3, or uponallelic or homologous genomic and/or cDNA sequences, one of skill in theart can easily choose and synthesize any of a number of appropriateantisense molecules for use in accordance with the present invention. Inorder to be sufficiently selective and potent for inhibition, suchantisense oligonucleotides should comprise at least 10 and, morepreferably, at least 15 consecutive bases which are complementary to thetarget, although in certain cases modified oligonucleotides as short as7 bases in length have been used successfully as antisenseoligonucleotides (Wagner et al., Nat. Med, 1995, 1(11):1116-1118; Nat.Biotech., 1996, 14:840-844). Most preferably, the antisenseoligonucleotides comprise a complementary sequence of 20-30 bases.

Although oligonucleotides may be chosen which are antisense to anyregion of the gene or mRNA transcripts, in preferred embodiments theantisense oligonucleotides correspond to N-terminal or 5′ upstream sitessuch as translation initiation, transcription initiation or promotersites. In addition, 3′-untranslated regions may be targeted by antisenseoligonucleotides. Targeting to mRNA splicing sites has also been used inthe art but may be less preferred if alternative mRNA splicing occurs.In addition, the antisense is targeted, preferably, to sites in whichmRNA secondary structure is not expected (see, e.g., Sainio et al., CellMol. Neurobiol. 14(5):439-457, 1994) and at which proteins are notexpected to bind. Finally, although, SEQ ID NOs: 1 and 3, disclose cDNAsequences, one of ordinary skill in the art may easily derive thegenomic DNA corresponding to the foregoing sequences. Thus, the presentinvention also provides for antisense oligonucleotides which arecomplementary to the genomic DNA corresponding to SEQ ID NOs: 1 and 3.Similarly, antisense to allelic or homologous human cDNAs and genomicDNAs are enabled without undue experimentation.

The oligonucleotides of the invention may include RNAi molecules. Theuse of RNA interference or “RNAi” involves the use of double-strandedRNA (dsRNA) to block gene expression. (see, e.g. Sui, G, et al, ProcNatl. Acad. Sci U.S.A. 99:5515-5520, 2002). Methods of applying RNAistrategies in embodiments of the invention will be known to one ofordinary skill in the art.

In one set of embodiments, the antisense oligonucleotides of theinvention may be composed of “natural” deoxyribonucleotides,ribonucleotides, or any combination thereof. That is, the 5′ end of onenative nucleotide and the 3′ end of another native nucleotide may becovalently linked, as in natural systems, via a phosphodiesterinternucleoside linkage. These oligonucleotides may be prepared by artrecognized methods which may be carried out manually or by an automatedsynthesizer. They also may be produced recombinantly by vectors.

In preferred embodiments, however, the antisense oligonucleotides of theinvention also may include “modified” oligonucleotides. That is, theoligonucleotides may be modified in a number of ways which do notprevent them from hybridizing to their target but which enhance theirstability or targeting or which otherwise enhance their therapeuticeffectiveness.

The term “modified oligonucleotide” as used herein describes anoligonucleotide in which (1) at least two of its nucleotides arecovalently linked via a synthetic internucleoside linkage (i.e., alinkage other than a phosphodiester linkage between the 5′ end of onenucleotide and the 3′ end of another nucleotide) and/or (2) a chemicalgroup not normally associated with nucleic acids has been covalentlyattached to the oligonucleotide. Preferred synthetic internucleosidelinkages are phosphorothioates, alkylphosphonates, phosphorodithioates,phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates,carbonates, phosphate triesters, acetamidates, carboxymethyl esters andpeptides.

The term “modified oligonucleotide” also encompasses oligonucleotideswith a covalently modified base and/or sugar. For example, modifiedoligonucleotides include oligonucleotides having backbone sugars whichare covalently attached to low molecular weight organic groups otherthan a hydroxyl group at the 3′ position and other than a phosphategroup at the 5′ position. Thus modified oligonucleotides may include a2′-O-alkylated ribose group. In addition, modified oligonucleotides mayinclude sugars such as arabinose in place of ribose. The presentinvention, thus, contemplates pharmaceutical preparations containingmodified antisense molecules that are complementary to and hybridizablewith, under physiological conditions, nucleic acids encoding thepolypeptides with SEQ ID NOs: 2, and/or 4, together withpharmaceutically acceptable carriers.

Antisense oligonucleotides may be administered as part of apharmaceutical composition. Such a pharmaceutical composition mayinclude the antisense oligonucleotides in combination with any standardphysiologically and/or pharmaceutically acceptable carriers which areknown in the art. The compositions should be sterile and contain atherapeutically effective amount of the antisense oligonucleotides in aunit of weight or volume suitable for administration to a patient. Theterm “pharmaceutically acceptable” means a non-toxic material that doesnot interfere with the effectiveness of the biological activity of theactive ingredients. The term “physiologically acceptable” refers to anon-toxic material that is compatible with a biological system such as acell, cell culture, tissue, or organism. The characteristics of thecarrier will depend on the route of administration. Physiologically andpharmaceutically acceptable carriers include diluents, fillers, salts,buffers, stabilizers, solubilizers, and other materials which are wellknown in the art.

The invention also involves expression vectors coding for proteinsencoded by the nucleic acids corresponding to SEQ ID NOs: 1 and/or 3,fragments and variants thereof, and host cells containing thoseexpression vectors. Virtually any cell, prokaryotic or eukaryotic, whichcan be transformed with heterologous DNA or RNA and which can be grownor maintained in culture, may be used in the practice of the invention.Examples include bacterial cells such as Escherichia coli and mammaliancells such as mouse, hamster, pig, goat, primate, etc. They may be of awide variety of tissue types, including mast cells, fibroblasts, oocytesand lymphocytes, and they may be primary cells or cell lines. Specificexamples include CHO cells and COS cells. Cell-free transcriptionsystems also may be used in lieu of cells.

As used herein, a “vector” may be any of a number of nucleic acids intowhich a desired sequence may be inserted by restriction and ligation fortransport between different genetic environments or for expression in ahost cell. Vectors are typically composed of DNA although RNA vectorsare also available. Vectors include, but are not limited to, plasmids,phagemids and virus genomes. A cloning vector is one which is able toreplicate in a host cell, and which is further characterized by one ormore endonuclease restriction sites at which the vector may be cut in adeterminable fashion and into which a desired DNA sequence may beligated such that the new recombinant vector retains its ability toreplicate in the host cell. In the case of plasmids, replication of thedesired sequence may occur many times as the plasmid increases in copynumber within the host bacterium or just a single time per host beforethe host reproduces by mitosis. In the case of phage, replication mayoccur actively during a lytic phase or passively during a lysogenicphase. An expression vector is one into which a desired DNA sequence maybe inserted by restriction and ligation such that it is operably joinedto regulatory sequences and may be expressed as an RNA transcript.Vectors may further contain one or more marker sequences suitable foruse in the identification of cells which have or have not beentransformed or transfected with the vector. Markers include, forexample, genes encoding proteins which increase or decrease eitherresistance or sensitivity to antibiotics or other compounds, genes whichencode enzymes whose activities are detectable by standard assays knownin the art (e.g., β-galactosidase or alkaline phosphatase), and geneswhich visibly affect the phenotype of transformed or transfected cells,hosts, colonies or plaques (e.g., green fluorescent protein). Preferredvectors are those capable of autonomous replication and expression ofthe structural gene products present in the DNA segments to which theyare operably joined.

As used herein, a coding sequence and regulatory sequences are said tobe “operably joined” when they are covalently linked in such a way as toplace the expression or transcription of the coding sequence under theinfluence or control of the regulatory sequences. If it is desired thatthe coding sequences be translated into a functional protein, two DNAsequences are said to be operably joined if induction of a promoter inthe 5′ regulatory sequences results in the transcription of the codingsequence and if the nature of the linkage between the two DNA sequencesdoes not (1) result in the introduction of a frame-shift mutation, (2)interfere with the ability of the promoter region to direct thetranscription of the coding sequences, or (3) interfere with the abilityof the corresponding RNA transcript to be translated into a protein.Thus, a promoter region would be operably joined to a coding sequence ifthe promoter region were capable of effecting transcription of that DNAsequence such that the resulting transcript might be translated into thedesired protein or polypeptide.

The precise nature of the regulatory sequences needed for geneexpression may vary between species or cell types, but shall in generalinclude, as necessary, 5′ non-transcribed and 5′ non-translatedsequences involved with the initiation of transcription and translationrespectively, such as a TATA box, capping sequence, CAAT sequence, andthe like. Such 5′ non-transcribed regulatory sequences will ofteninclude a promoter region which includes a promoter sequence fortranscriptional control of the operably joined gene. Regulatorysequences may also include enhancer sequences or upstream activatorsequences as desired. The vectors of the invention may optionallyinclude 5′ leader or signal sequences. The choice and design of anappropriate vector is within the ability and discretion of one ofordinary skill in the art.

Expression vectors containing all the necessary elements for expressionare commercially available and known to those skilled in the art. See,e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, 1989. Cells aregenetically engineered by the introduction into the cells ofheterologous DNA (RNA) encoding a polypeptide or fragment or variantthereof. That heterologous DNA (RNA) is placed under operable control oftranscriptional elements to permit the expression of the heterologousDNA in the host cell.

Preferred systems for mRNA expression in mammalian cells are those suchas pcDNA3.1 (available from Invitrogen, Carlsbad, Calif.) that contain aselectable marker such as a gene that confers G418 resistance (whichfacilitates the selection of stably transfected cell lines) and thehuman cytomegalovirus (CMV) enhancer-promoter sequences. Additionally,suitable for expression in primate or canine cell lines is the pCEP4vector (Invitrogen, Carlsbad, Calif.), which contains an Epstein Barrvirus (EBV) origin of replication, facilitating the maintenance ofplasmid as a multicopy extrachromosomal element. Still another preferredexpression vector is an adenovirus, described by Stratford-Perricaudet,which is defective for E1 and E3 proteins (J. Clin. Invest. 90:626-630,1992).

The invention also embraces so-called expression kits, which allow theartisan to prepare a desired expression vector or vectors. Suchexpression kits include at least separate portions of each of thepreviously discussed coding sequences. Other components may be added, asdesired, as long as the previously mentioned sequences, which arerequired, are included.

It will also be recognized that the invention embraces the use of theabove described SEQ ID NOs: 1 and/or 3, cDNA sequence-containingexpression vectors, to transfect host cells and cell lines, be theseprokaryotic (e.g., Escherichia coli), or eukaryotic (e.g., CHO cells,COS cells, yeast expression systems and recombinant baculovirusexpression in insect cells). Especially useful are mammalian cells suchas mouse, hamster, pig, goat, primate, etc. They may be of a widevariety of tissue types, and include primary cells and cell lines.Specific examples include dendritic cells, U293 cells, peripheral bloodleukocytes, bone marrow stem cells and embryonic stem cells.

The invention also provides isolated polypeptides (including wholeproteins and partial proteins), encoded by the foregoing nucleic acids(SEQ ID NOs: 1 and 3), and include the polypeptides of SEQ ID NOs: 2and/or 4, and unique fragments thereof. Such polypeptides are useful,for example, alone or as part of fusion proteins to generate antibodies,as components of an immunoassay, etc. Polypeptides can be isolated frombiological samples including tissue or cell homogenates, and can also beexpressed recombinantly in a variety of prokaryotic and eukaryoticexpression systems by constructing an expression vector appropriate tothe expression system, introducing the expression vector into theexpression system, and isolating the recombinantly expressed protein.Short polypeptides, including antigenic peptides (such as are presentedby MHC molecules on the surface of a cell for immune recognition) alsocan be synthesized chemically using well-established methods of peptidesynthesis.

As used herein with respect to polypeptides, the term “isolated” meansseparated from its native environment in sufficiently pure form so thatit can be manipulated or used for any one of the purposes of theinvention. Thus, isolated means sufficiently pure to be used (i) toraise and/or isolate antibodies, (ii) as a reagent in an assay, (iii)for sequencing, (iv) as a therapeutic, etc.

A unique fragment for each of the foregoing polypeptide, in general, hasthe features and characteristics of unique fragments as discussed abovein connection with nucleic acids. As will be recognized by those skilledin the art, the size of the unique fragment will depend upon factorssuch as whether the fragment constitutes a portion of a conservedprotein domain. Thus, some regions of a polypeptide will require longersegments to be unique while others will require only short segments,typically between 5 and 12 amino acids (e.g. 5, 6, 7, 8, 9, 10, 11 and12 amino acids long or more, including each integer up to the fulllength of each polypeptide).

Unique fragments of a polypeptide preferably are those fragments whichretain a distinct functional capability of the polypeptide. Functionalcapabilities which can be retained in a unique fragment of a polypeptideinclude interaction with antibodies, interaction with other polypeptidesor fragments thereof, interaction with other molecules, etc. Oneimportant activity is the ability to act as a signature for identifyingthe polypeptide. Those skilled in the art are well versed in methods forselecting unique amino acid sequences, typically on the basis of theability of the unique fragment to selectively distinguish the sequenceof interest from non-family members. A comparison of the sequence of thefragment to those on known databases typically is all that is necessary.

The invention embraces variants of the polypeptides described above. Asused herein, a “variant” of a polypeptide is a polypeptide whichcontains one or more modifications to the primary amino acid sequence ofa natural (e.g., “wild-type”: a polypeptide with an amino acid sequenceselected from the group consisting of SEQ ID NO: 2 and 4) polypeptide.Modifications which create a polypeptide variant are typically made tothe nucleic acid which encodes the polypeptide, and can includedeletions, point mutations, truncations, amino acid substitutions andaddition of amino acids or non-amino acid moieties to: (1) reduce oreliminate an activity of a polypeptide; (2) enhance a property of apolypeptide, such as protein stability in an expression system or thestability of protein-ligand binding; (3) provide a novel activity orproperty to a polypeptide, such as addition of an antigenic epitope oraddition of a detectable moiety; or (4) to provide equivalent or betterbinding to a polypeptide receptor or other molecule. Alternatively,modifications can be made directly to the polypeptide, such as bycleavage, addition of a linker molecule, addition of a detectablemoiety, such as biotin, addition of a fatty acid, and the like.Modifications also embrace fusion proteins comprising all or part of thepolypeptide's amino acid sequence. One of skill in the art will befamiliar with methods for predicting the effect on protein conformationof a change in protein sequence, and can thus “design” a variantpolypeptide according to known methods. One example of such a method isdescribed by Dahiyat and Mayo in Science 278:82-87, 1997, wherebyproteins can be designed de novo. The method can be applied to a knownprotein to vary only a portion of the polypeptide sequence. By applyingthe computational methods of Dahiyat and Mayo, specific variants of anyof the foregoing polypeptides can be proposed and tested to determinewhether the variant retains a desired conformation.

Variants can include polypeptides which are modified specifically toalter a feature of the polypeptide unrelated to its physiologicalactivity. For example, cysteine residues can be substituted or deletedto prevent unwanted disulfide linkages. Similarly, certain amino acidscan be changed to enhance expression of a polypeptide by eliminatingproteolysis by proteases in an expression system (e.g., dibasic aminoacid residues in yeast expression systems in which KEX2 proteaseactivity is present).

Mutations of a nucleic acid which encodes a polypeptide preferablypreserve the amino acid reading frame of the coding sequence, andpreferably do not create regions in the nucleic acid which are likely tohybridize to form secondary structures, such as hairpins or loops, whichcan be deleterious to expression of the variant polypeptide.

Mutations can be made by selecting an amino acid substitution, or byrandom mutagenesis of a selected site in a nucleic acid which encodesthe polypeptide. Variant polypeptides are then expressed and tested forone or more activities to determine which mutation provides a variantpolypeptide with the desired properties. Further mutations can be madeto variants (or to non-variant polypeptides) which are silent as to theamino acid sequence of the polypeptide, but which provide preferredcodons for translation in a particular host. The preferred codons fortranslation of a nucleic acid in, e.g., Escherichia coli, are well knownto those of ordinary skill in the art. Still other mutations can be madeto the noncoding sequences of a gene or cDNA clone to enhance expressionof the polypeptide.

The skilled artisan will realize that conservative amino acidsubstitutions may be made in any of the foregoing polypeptides toprovide functionally equivalent variants of the foregoing polypeptides,i.e., the variants retain the functional capabilities of eachpolypeptide. As used herein, a “conservative amino acid substitution”refers to an amino acid substitution which does not significantly alterthe tertiary structure and/or activity of the polypeptide. Variants canbe prepared according to methods for altering polypeptide sequence knownto one of ordinary skill in the art, and include those that are found inreferences which compile such methods, e.g. Molecular Cloning: ALaboratory Manual, J. Sambrook, et al., eds., Second Edition, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, orCurrent Protocols in Molecular Biology, F. M. Ausubel, et al., eds.,John Wiley & Sons, Inc., New York. Conservative substitutions of aminoacids include substitutions made amongst amino acids within thefollowing groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G;(e) S, T; (f) Q, N; and (g) E, D.

Thus functionally equivalent variants of polypeptides, i.e., variants ofpolypeptides which retain the function of the natural (“wild-type”)polypeptides, are contemplated by the invention. Conservative amino acidsubstitutions in the amino acid sequence of polypeptides to producefunctionally equivalent variants of each polypeptide typically are madeby alteration of a nucleic acid encoding the polypeptide. Suchsubstitutions can be made by a variety of methods known to one ofordinary skill in the art. For example, amino acid substitutions may bemade by PCR-directed mutation, site-directed mutagenesis according tothe method of Kunkel (Kunkel, Proc. Nat. Acad. Sci. U.S.A. 82: 488-492,1985), or by chemical synthesis of a gene encoding a polypeptide. Theactivity of functionally equivalent fragments of polypeptides can betested by cloning the gene encoding the altered polypeptide into abacterial or mammalian expression vector, introducing the vector into anappropriate host cell, expressing the altered polypeptide, and testingfor a functional capability of the polypeptides as disclosed herein

The invention as described herein has a number of uses, some of whichare described elsewhere herein. First, the invention permits isolationof polypeptides. A variety of methodologies well-known to the skilledartisan can be utilized to obtain isolated molecules. The polypeptidemay be purified from cells which naturally produce the polypeptide bychromatographic means or immunological recognition. Alternatively, anexpression vector may be introduced into cells to cause production ofthe polypeptide. In another method, mRNA transcripts may bemicroinjected or otherwise introduced into cells to cause production ofthe encoded polypeptide. Translation of mRNA in cell-free extracts suchas the reticulocyte lysate system also may be used to producepolypeptides. Those skilled in the art also can readily follow knownmethods for isolating polypeptides. These include, but are not limitedto, immunochromatography, HPLC, size-exclusion chromatography,ion-exchange chromatography and immune-affinity chromatography.

The invention also provides, in certain embodiments, “dominant negative”polypeptides derived from polypeptides. A dominant negative polypeptideis an inactive variant of a protein, which, by interacting with thecellular machinery, displaces an active protein from its interactionwith the cellular machinery or competes with the active protein, therebyreducing the effect of the active protein. For example, a dominantnegative receptor which binds a ligand but does not transmit a signal inresponse to binding of the ligand can reduce the biological effect ofexpression of the ligand. Likewise, a dominant negativecatalytically-inactive kinase which interacts normally with targetproteins but does not phosphorylate the target proteins can reducephosphorylation of the target proteins in response to a cellular signal.Similarly, a dominant negative transcription factor which binds to apromoter site in the control region of a gene but does not increase genetranscription can reduce the effect of a normal transcription factor byoccupying promoter binding sites without increasing transcription.

The end result of the expression of a dominant negative polypeptide in acell is a reduction in function of active proteins. One of ordinaryskill in the art can assess the potential for a dominant negativevariant of a protein, and use standard mutagenesis techniques to createone or more dominant negative variant polypeptides. See, e.g., U.S. Pat.No. 5,580,723 and Sambrook et al., Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Theskilled artisan then can test the population of mutagenized polypeptidesfor diminution in a selected activity and/or for retention of such anactivity. Other similar methods for creating and testing dominantnegative variants of a protein will be apparent to one of ordinary skillin the art.

The isolation of the cDNAs of the invention also makes it possible forthe artisan to diagnose a disorder characterized by an aberrantexpression of any of the foregoing cDNAs. These methods involvedetermining expression of each of the identified nucleic acids, and/orpolypeptides derived therefrom. In the former situation, suchdeterminations can be carried out via any standard nucleic aciddetermination assay, including the polymerase chain reaction, orassaying with labeled hybridization probes as exemplified below. In thelatter situation, such determination can be carried out via any standardimmunological assay using, for example, antibodies which bind to thesecreted protein.

The invention also embraces isolated peptide binding agents which, forexample, can be antibodies or fragments of antibodies (“bindingpolypeptides”), having the ability to selectively bind to any of thepolypeptides of the invention (e.g., SEQ ID NO: 2 or 4). Antibodiesinclude polyclonal and monoclonal antibodies, prepared according toconventional methodology.

Significantly, as is well-known in the art, only a small portion of anantibody molecule, the paratope, is involved in the binding of theantibody to its epitope (see, in general, Clark, W. R. (1986) TheExperimental Foundations of Modern Immunology Wiley & Sons, Inc., NewYork; Roitt, I. (1991) Essential Immunology, 7th Ed., BlackwellScientific Publications, Oxford). The pFc′ and Fc regions, for example,are effectors of the complement cascade but are not involved in antigenbinding. An antibody from which the pFc′ region has been enzymaticallycleaved, or which has been produced without the pFc′ region, designatedan F(ab′)₂ fragment, retains both of the antigen binding sites of anintact antibody. Similarly, an antibody from which the Fc region hasbeen enzymatically cleaved, or which has been produced without the Fcregion, designated an Fab fragment, retains one of the antigen bindingsites of an intact antibody molecule. Proceeding further, Fab fragmentsconsist of a covalently bound antibody light chain and a portion of theantibody heavy chain denoted Fd. The Fd fragments are the majordeterminant of antibody specificity (a single Fd fragment may beassociated with up to ten different light chains without alteringantibody specificity) and Fd fragments retain epitope-binding ability inisolation.

Within the antigen-binding portion of an antibody, as is well-known inthe art, there are complementarity determining regions (CDRs), whichdirectly interact with the epitope of the antigen, and framework regions(FRs), which maintain the tertiary structure of the paratope (see, ingeneral, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragmentand the light chain of IgG immunoglobulins, there are four frameworkregions (FR1 through FR4) separated respectively by threecomplementarity determining regions (CDR1 through CDR3). The CDRs, andin particular the CDR3 regions, and more particularly the heavy chainCDR3, are largely responsible for antibody specificity.

It is now well-established in the art that the non-CDR regions of amammalian antibody may be replaced with similar regions of conspecificor heterospecific antibodies while retaining the epitopic specificity ofthe original antibody. This is most clearly manifested in thedevelopment and use of “humanized” antibodies in which non-human CDRsare covalently joined to human FR and/or Fc/pFc′ regions to produce afunctional antibody. See, e.g., U.S. Pat. Nos. 4,816,567; 5,225,539;5,585,089; 5,693,762 and 5,859,205. Thus, for example, PCT InternationalPublication Number WO 92/04381 teaches the production and use ofhumanized murine RSV antibodies in which at least a portion of themurine FR regions have been replaced by FR regions of human origin. Suchantibodies, including fragments of intact antibodies withantigen-binding ability, are often referred to as “chimeric” antibodies.

Thus, as will be apparent to one of ordinary skill in the art, thepresent invention also provides for F(ab′)₂, Fab, Fv and Fd fragments;chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2and/or light chain CDR3 regions have been replaced by homologous humanor non-human sequences; chimeric F(ab′)₂ fragment antibodies in whichthe FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have beenreplaced by homologous human or non-human sequences; chimeric Fabfragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or lightchain CDR3 regions have been replaced by homologous human or non-humansequences; and chimeric Fd fragment antibodies in which the FR and/orCDR1 and/or CDR2 regions have been replaced by homologous human ornon-human sequences. The present invention also includes so-calledsingle chain antibodies.

Thus, the invention involves polypeptides of numerous size and type thatbind specifically to polypeptides of the invention (e.g., SEQ ID NO: 2,or 4-its extracellular portions), and complexes of both the polypeptidesand their binding partners. These polypeptides may be derived also fromsources other than antibody technology. For example, such polypeptidebinding agents can be provided by degenerate peptide libraries which canbe readily prepared in solution, in immobilized form, as bacterialflagella peptide display libraries or as phage display libraries.Combinatorial libraries also can be synthesized of peptides containingone or more amino acids. Libraries further can be synthesized ofpeptides and non-peptide synthetic moieties.

The invention further provides efficient methods of identifying agentsor lead compounds for agents active at the level of a polypeptide orpolypeptide fragment dependent cellular function. In particular, suchfunctions include interaction with other polypeptides or fragments.Generally, the screening methods involve assaying for compounds whichinterfere with the activity of a polypeptide of the invention, althoughcompounds which enhance such activity also can be assayed using thescreening methods. Such methods are adaptable to automated, highthroughput screening of compounds. Target indications include cellularprocesses modulated by such polypeptides, for example, overexpression incells under mechanical strains.

A wide variety of assays for candidate (pharmacological) agents areprovided, including, labeled in vitro protein-ligand binding assays,electrophoretic mobility shift assays, immunoassays, cell-based assayssuch as two- or three-hybrid screens, expression assays, etc. Thetransfected nucleic acids can encode, for example, combinatorial peptidelibraries or cDNA libraries. Convenient reagents for such assays, e.g.,GAL4 fusion proteins, are known in the art. An exemplary cell-basedassay involves transfecting a cell with a nucleic acid encoding apolypeptide of the invention fused to a GAL4 DNA binding domain and anucleic acid encoding a reporter gene operably joined to a geneexpression regulatory region, such as one or more GAL4 binding sites.Activation of reporter gene transcription occurs when the reporterfusion polypeptide binds an agent such as to enable transcription of thereporter gene. Agents which modulate polypeptide mediated cell functionare then detected through a change in the expression of reporter gene.Methods for determining changes in the expression of a reporter gene areknown in the art.

Polypeptide fragments used in the methods, when not produced by atransfected nucleic acid are added to an assay mixture as an isolatedpolypeptide. Polypeptides preferably are produced recombinantly,although such polypeptides may be isolated from biological extracts.Recombinantly produced polypeptides include chimeric proteins comprisinga fusion of a protein of the invention with another polypeptide, e.g., apolypeptide capable of providing or enhancing protein-protein binding,sequence specific nucleic acid binding (such as GAL4), enhancingstability of the polypeptide of the invention under assay conditions, orproviding a detectable moiety, such as green fluorescent protein or aFlag epitope.

The assay mixture is comprised of a natural intracellular orextracellular binding target capable of interacting with a polypeptideof the invention. While natural polypeptide binding targets may be used,it is frequently preferred to use portions (e.g., peptides or nucleicacid fragments) or analogs (i.e., agents which mimic the polypeptide'sbinding properties of the natural binding target for purposes of theassay) of the polypeptide binding target so long as the portion oranalog provides binding affinity and avidity to the polypeptide fragmentmeasurable in the assay.

The assay mixture also comprises a candidate agent. Typically, aplurality of assay mixtures are run in parallel with different agentconcentrations to obtain a different response to the variousconcentrations. Typically, one of these concentrations serves as anegative control, i.e., at zero concentration of agent or at aconcentration of agent below the limits of assay detection. Candidateagents encompass numerous chemical classes, although typically they areorganic compounds. Preferably, the candidate agents are small organiccompounds, i.e., those having a molecular weight of more than about 50yet less than about 2500, preferably less than about 1000 and, morepreferably, less than about 500. Candidate agents comprise functionalchemical groups necessary for structural interactions with polypeptidesand/or nucleic acids, and typically include at least an amine, carbonyl,hydroxyl or carboxyl group, preferably at least two of the functionalchemical groups and more preferably at least three of the functionalchemical groups. The candidate agents can comprise cyclic carbon orheterocyclic structure and/or aromatic or polyaromatic structuressubstituted with one or more of the above-identified functional groups.Candidate agents also can be biomolecules such as peptides, saccharides,fatty acids, sterols, isoprenoids, purines, pyrimidines, derivatives orstructural analogs of the above, or combinations thereof and the like.Where the agent is a nucleic acid, the agent typically is a DNA or RNAmolecule, although modified nucleic acids as defined herein are alsocontemplated.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides, synthetic organic combinatorial libraries, phagedisplay libraries of random peptides, and the like. Alternatively,libraries of natural compounds in the form of bacterial, fungal, plantand animal extracts are available or readily produced. Additionally,natural and synthetically produced libraries and compounds can bemodified through conventional chemical, physical, and biochemical means.Further, known (pharmacological) agents may be subjected to directed orrandom chemical modifications such as acylation, alkylation,esterification, amidification, etc. to produce structural analogs of theagents.

A variety of other reagents also can be included in the mixture. Theseinclude reagents such as salts, buffers, neutral proteins (e.g.,albumin), detergents, etc. which may be used to facilitate optimalprotein-protein and/or protein-nucleic acid binding. Such a reagent mayalso reduce non-specific or background interactions of the reactioncomponents. Other reagents that improve the efficiency of the assay suchas protease inhibitors, nuclease inhibitors, antimicrobial agents, andthe like may also be used.

The mixture of the foregoing assay materials is incubated underconditions whereby, but for the presence of the candidate agent, thechosen polypeptide of the invention specifically binds a cellularbinding target, a portion thereof or analog thereof. The order ofaddition of components, incubation temperature, time of incubation, andother parameters of the assay may be readily determined. Suchexperimentation merely involves optimization of the assay parameters,not the fundamental composition of the assay. Incubation temperaturestypically are between 4° C. and 40° C. Incubation times preferably areminimized to facilitate rapid, high throughput screening, and typicallyare between 0.1 and 10 hours.

After incubation, the presence or absence of specific binding betweenthe polypeptide and one or more binding targets is detected by anyconvenient method available to the user. For cell free binding typeassays, a separation step is often used to separate bound from unboundcomponents. The separation step may be accomplished in a variety ofways. Conveniently, at least one of the components is immobilized on asolid substrate, from which the unbound components may be easilyseparated. The solid substrate can be made of a wide variety ofmaterials and in a wide variety of shapes, e.g., microtiter plate,microbead, dipstick, resin particle, etc. The substrate preferably ischosen to maximize signal to noise ratios, primarily to minimizebackground binding, as well as for ease of separation and cost.

Separation may be effected for example, by removing a bead or dipstickfrom a reservoir, emptying or diluting a reservoir such as a microtiterplate well, rinsing a bead, particle, chromatographic column or filterwith a wash solution or solvent. The separation step preferably includesmultiple rinses or washes. For example, when the solid substrate is amicrotiter plate, the wells may be washed several times with a washingsolution, which typically includes those components of the incubationmixture that do not participate in specific bindings such as salts,buffer, detergent, a non-specific protein, etc. When the solid substrateis a magnetic bead(s), the bead(s) may be washed one or more times witha washing solution and isolated using a magnet.

Detection may be effected in any convenient way for cell-based assayssuch as two- or three-hybrid screens. The transcript resulting from areporter gene transcription assay of a polypeptide interacting with atarget molecule typically encodes a directly or indirectly detectableproduct, e.g., β-galactosidase activity, luciferase activity, and thelike. For cell free binding assays, one of the components usuallycomprises, or is coupled to, a detectable label. A wide variety oflabels can be used, such as those that provide direct detection (e.g.,radioactivity, luminescence, optical or electron density, etc), orindirect detection (e.g., epitope tag such as the FLAG epitope, enzymetag such as horseradish peroxidase, etc.). The label may be bound to abinding partner of the polypeptide, or incorporated into the structureof the binding partner.

A variety of methods may be used to detect the label, depending on thenature of the label and other assay components. For example, the labelmay be detected while bound to the solid substrate or subsequent toseparation from the solid substrate. Labels may be directly detectedthrough optical or electron density, radioactive emissions, nonradiativeenergy transfers, etc. or indirectly detected with antibody conjugates,streptavidin-biotin conjugates, etc. Methods for detecting the labelsare well known in the art.

The invention provides polypeptide-specific binding agents, methods ofidentifying and making such agents, and their use in diagnosis, therapyand pharmaceutical development. For example, polypeptide-specificpharmacological agents are useful in a variety of diagnostic andtherapeutic applications, especially where disease or disease prognosisis associated with altered polypeptide binding characteristics. Novelpolypeptide-specific binding agents include polypeptide-specificantibodies, cell surface receptors, and other natural intracellular andextracellular binding agents identified with assays such as two hybridscreens, and non-natural intracellular and extracellular binding agentsidentified in screens of chemical libraries and the like.

In general, the specificity of polypeptide binding to a specificmolecule is determined by binding equilibrium constants. Targets whichare capable of selectively binding a polypeptide preferably have bindingequilibrium constants of at least about 10⁷ M⁻¹, more preferably atleast about 10⁸ M⁻¹, and most preferably at least about 10⁹ M⁻¹. A widevariety of cell based and cell free assays may be used to demonstratepolypeptide-specific binding. Cell based assays include one, two andthree hybrid screens, assays in which polypeptide-mediated transcriptionis inhibited or increased, etc. Cell free assays include protein bindingassays, immunoassays, etc. Other assays useful for screening agentswhich bind polypeptides of the invention include fluorescence resonanceenergy transfer (FRET), and electrophoretic mobility shift analysis(EMSA).

According to still another aspect of the invention, a method ofdiagnosing a disorder characterized by aberrant expression of a nucleicacid molecule, an expression product thereof, or a fragment of anexpression product thereof, is provided. The method involves contactinga biological sample isolated from a subject with an agent thatspecifically binds to the nucleic acid molecule, an expression productthereof, or a fragment of an expression product thereof, and determiningthe interaction between the agent and the nucleic acid molecule or theexpression product as a determination of the disorder, wherein thenucleic acid molecule is a IL1RL-1 nucleic acid (SEQ ID NO.:1). In someembodiments, the disorder is a cardiovascular condition selected fromthe group consisting of myocardial infarction, stroke, arteriosclerosis,and heart failure. In one embodiment, the disorder is cardiachypertrophy. In another embodiment, the disorder is myocardialinfarction. In one embodiment, the disorder is heart failure.

In the case where the molecule is a nucleic acid molecule, suchdeterminations can be carried out via any standard nucleic aciddetermination assay, including the polymerase chain reaction, orassaying with labeled hybridization probes as exemplified herein. In thecase where the molecule is an expression product of the nucleic acidmolecule, or a fragment of an expression product of the nucleic acidmolecule, such determination can be carried out via any standardimmunological assay using, for example, antibodies which bind to any ofthe polypeptide expression products.

“Aberrant expression” refers to decreased expression (underexpression)or increased expression (overexpression) of any of the foregoing IL1RL-1molecules (nucleic acids and/or polypeptides) in comparison with acontrol (i.e., expression of the same molecule in a healthy or “normal”subject). A “healthy subject,” as used herein, refers to a subject whois not at risk for developing a future cardiovascular condition (seeearlier discussion and Harrison's Principles of Experimental Medicine,13th Edition, McGraw-Hill, Inc., N.Y.—hereinafter “Harrison's”). Healthysubjects also do not otherwise exhibit symptoms of disease. In otherwords, such subjects, if examined by a medical professional, would becharacterized as healthy and free of symptoms of a cardiovasculardisorder or at risk of developing a cardiovascular disorder.

When the disorder is a cardiovascular condition selected from the groupconsisting of cardiac hypertrophy, myocardial infarction, stroke,arteriosclerosis, and heart failure, decreased expression of any of theforegoing molecules in comparison with a control (e.g., a healthysubject) is indicative of the presence of the disorder, or indicative ofthe risk for developing such disorder in the future.

The invention also provides novel kits which could be used to measurethe levels of the nucleic acids of the invention, or expression productsof the invention.

In one embodiment, a kit comprises a package containing an agent thatselectively binds to an isolated IL1RL-1 nucleic acid, or expressionproduct thereof, and a control for comparing to a measured value ofbinding of said agent any of the foregoing isolated nucleic acids orexpression products thereof. Kits are generally comprised of thefollowing major elements: packaging, an agent of the invention, acontrol agent, and instructions. Packaging may be a box-like structurefor holding a vial (or number of vials) containing an agent of theinvention, a vial (or number of vials) containing a control agent, andinstructions. Individuals skilled in the art can readily modify thepackaging to suit individual needs. In some embodiments, the control isa predetermined value for comparing to the measured value. In certainembodiments, the control comprises an epitope of the expression productof any of the foregoing isolated nucleic acids.

In the case of nucleic acid detection, pairs of primers for amplifying anucleic acid molecule of the invention can be included. The preferredkits would include known amounts of nucleic acid probes, epitopes (suchas IL1RL-1 expression products) or anti-epitope antibodies, as well asinstructions or other printed material. In certain embodiments theprinted material can characterize risk of developing a cardiovascularcondition based upon the outcome of the assay. The reagents may bepackaged in containers and/or coated on wells in predetermined amounts,and the kits may include standard materials such as labeledimmunological reagents (such as labeled anti-IgG antibodies) and thelike. One kit is a packaged polystyrene microtiter plate coated with anyof the foregoing proteins of the invention and a container containinglabeled anti-human IgG antibodies. A well of the plate is contactedwith, for example, a biological fluid, washed and then contacted withthe anti-IgG antibody. The label is then detected. A kit embodyingfeatures of the present invention, generally designated by the numeral11, is illustrated in FIG. 7. Kit 11 is comprised of the following majorelements: packaging 15, an agent of the invention 17, a control agent19, and instructions 21. Packaging 15 is a box-like structure forholding a vial (or number of vials) containing an agent of the invention17, a vial (or number of vials) containing a control agent 19, andinstructions 21. Individuals skilled in the art can readily modifypackaging 15 to suit individual needs.

In preferred embodiments the invention provides novel kits or assayswhich are specific for, and have appropriate sensitivity with respectto, predetermined values selected on the basis of the present invention.The preferred kits, therefore, would differ from those presentlycommercially available, by including, for example, different cut-offs,different sensitivities at particular cut-offs as well as instructionsor other printed material for characterizing risk based upon the outcomeof the assay.

The invention also embraces methods for evaluating the likelihood that asubject will benefit from treatment with an agent for reducing the riskof a cardiovascular condition. In some embodiments the agent is selectedfrom the group consisting of an anti-inflammatory agent, anantithrombotic agent, an anti-platelet agent, a fibrinolytic agent, alipid reducing agent, a direct thrombin inhibitor, a glycoproteinIIb/IIIa receptor inhibitor, an agent that binds to cellular adhesionmolecules and inhibits the ability of white blood cells to attach tosuch molecules, a calcium channel blocker, a beta-adrenergic receptorblocker, a cyclooxygenase-2 inhibitor, and an angiotensin systeminhibitor. The method involves obtaining a level of a IL1RL-1 moleculein the subject, and comparing the level of the IL1RL-1 molecule to apredetermined value specific for the diagnosis of a cardiovascularcondition. The level of the IL1RL-1 molecule in comparison to thepredetermined value is indicative of whether the subject will benefitfrom treatment with said agent. In certain embodiments, thepredetermined value specific for the diagnosis of a cardiovascularcondition is a plurality of predetermined marker level ranges and saidcomparing step comprises determining in which of said predeterminedmarker level ranges said subjects level falls. The cardiovascularcondition can be a condition selected from the group consisting ofcardiac hypertrophy, myocardial infarction, stroke, arteriosclerosis,and heart failure.

The predetermined value can take a variety of forms. It can be singlecut-off value, such as a median or mean. It can be established basedupon comparative groups, such as where the risk in one defined group isdouble the risk in another defined group. It can be a range, forexample, where the tested population is divided equally (or unequally)into groups, such as a low-risk group, a medium-risk group and ahigh-risk group, or into quadrants, the lowest quadrant being subjectswith the lowest risk and the highest quadrant being subjects with thehighest risk.

The predetermined value can depend upon the particular populationselected. For example, an apparently healthy population (no detectabledisease and no prior history of a cardiovascular disorder) will have adifferent ‘normal’ range of markers of systemic inflammation than will asmoking population or a population the members of which have had a priorcardiovascular disorder. Accordingly, the predetermined values selectedmay take into account the category in which the subject falls.Appropriate ranges and categories can be selected with no more thanroutine experimentation by those of ordinary skill in the art.

As discussed above the invention provides methods for evaluating thelikelihood that a subject will benefit from treatment with an agent forreducing risk of a future cardiovascular disorder. This method hasimportant implications for patient treatment and also for clinicaldevelopment of new therapeutics. Physicians select therapeutic regimensfor patient treatment based upon the expected net benefit to thepatient. The net benefit is derived from the risk to benefit ratio. Thepresent invention permits selection of subjects who are more likely tobenefit by intervention, thereby aiding the physician in selecting atherapeutic regimen. This might include using drugs with a higher riskprofile where the likelihood of expected benefit has increased.Likewise, clinical investigators desire to select for clinical trials apopulation with a high likelihood of obtaining a net benefit. Thepresent invention can help clinical investigators select such subjects.It is expected that clinical investigators now will use the presentinvention for determining entry criteria for clinical trials.

The invention also embraces methods for treating a cardiovascularcondition. In some embodiments, the method involves administering to asubject in need of such treatment a IL1RL-1 molecule, in an amounteffective to treat the cardiovascular condition. In certain embodiments,the method involves administering to a subject in need of such treatmentan agent that modulate the expression of any of the foregoing Il1Rl-1molecules. “Agents that modulates expression” include any of the IL1RL-1molecules described herein, agents that increase expression of thesemolecules, as well as agents that decrease expression of any of theforegoing IL1RL-1 molecules, in an amount effective to treat thecardiovascular condition.

“Agents that decrease expression” of a nucleic acid or a polypeptide, asused herein, are known in the art, and refer to antisense nucleic acids,antibodies that bind polypeptides encoded by the nucleic acids, andother agents that lower expression of such molecules. Any agents thatdecrease expression of a molecule (and as described herein, decrease itsactivity), are useful according to the invention.

In certain embodiments, the molecule is a nucleic acid (antisense). Insome embodiments the nucleic acid is operatively coupled to a geneexpression sequence which directs the expression of the nucleic acidmolecule within a cardiomyocyte. The “gene expression sequence” is anyregulatory nucleotide sequence, such as a promoter sequence orpromoter-enhancer combination, which facilitates the efficienttranscription and translation of the nucleic acid to which it isoperably joined. The gene expression sequence may, for example, be amammalian or viral promoter, such as a constitutive or induciblepromoter. Constitutive mammalian promoters include, but are not limitedto, the promoters for the following genes: hypoxanthine phosphoribosyltransferase (HPTR), adenosine deaminase, pyruvate kinase, α-actinpromoter and other constitutive promoters. Exemplary viral promoterswhich function constitutively in eukaryotic cells include, for example,promoters from the simian virus, papilloma virus, adenovirus, humanimmunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus, thelong terminal repeats (LTR) of Moloney leukemia virus and otherretroviruses, and the thymidine kinase promoter of herpes simplex virus.Other constitutive promoters are known to those of ordinary skill in theart. The promoters useful as gene expression sequences of the inventionalso include inducible promoters. Inducible promoters are activated inthe presence of an inducing agent. For example, the metallothioneinpromoter is activated to increase transcription and translation in thepresence of certain metal ions. Other inducible promoters are known tothose of ordinary skill in the art.

In general, the gene expression sequence shall include, as necessary, 5′non-transcribing and 5′ non-translating sequences involved with theinitiation of transcription and translation, respectively, such as aTATA box, capping sequence, CAAT sequence, and the like. Especially,such 5′ non-transcribing sequences will include a promoter region whichincludes a promoter sequence for transcriptional control of the operablyjoined nucleic acid. The gene expression sequences optionally includesenhancer sequences or upstream activator sequences as desired.

Preferably, any of the IL1RL-1 nucleic acid molecules of the inventionis linked to a gene expression sequence which permits expression of thenucleic acid molecule in a cell such as a cardiomyocyte and/or avascular endothelial cell (including a smooth muscle cell). Morepreferably, the gene expression sequence permits expression of thenucleic acid molecule in a cardiomyocyte, and does not permit expressionof the molecule in a cell selected from the group consisting of aneuronal cell, a fibroblast, and a cell of hematopoietic origin. Asequence which permits expression of the nucleic acid molecule in acardiomyocyte, is one which is selectively active in such a cell type,thereby causing expression of the nucleic acid molecule in the cell. Thecardiac myosin heavy chain gene promoter, for example, can be used toexpress any of the foregoing nucleic acid molecules of the invention ina cardiomyocyte. Those of ordinary skill in the art will be able toeasily identify alternative promoters that are capable of expressing anucleic acid molecule in a cardiomyocyte.

The nucleic acid sequence and the gene expression sequence are said tobe “operably joined” when they are covalently linked in such a way as toplace the transcription and/or translation of the nucleic acid codingsequence under the influence or control of the gene expression sequence.If it is desired that the nucleic acid sequence be translated into afunctional protein, two DNA sequences are said to be operably joined ifinduction of a promoter in the 5′ gene expression sequence results inthe transcription of the nucleic acid sequence and if the nature of thelinkage between the two DNA sequences does not (1) result in theintroduction of a frame-shift mutation, (2) interfere with the abilityof the promoter region to direct the transcription of the nucleic acidsequence, and/or (3) interfere with the ability of the corresponding RNAtranscript to be translated into a protein. Thus, a gene expressionsequence would be operably linked to a nucleic acid sequence if the geneexpression sequence were capable of effecting transcription of thatnucleic acid sequence such that the resulting transcript might betranslated into the desired protein or polypeptide.

The molecules of the invention can be delivered to the preferred celltypes of the invention alone or in association with a vector. In itsbroadest sense, a “vector” is any vehicle capable of facilitating: (1)delivery of a molecule to a target cell and/or (2) uptake of themolecule by a target cell. Preferably, the vectors transport themolecule into the target cell with reduced degradation relative to theextent of degradation that would result in the absence of the vector.Optionally, a “targeting ligand” can be attached to the vector toselectively deliver the vector to a cell which expresses on its surfacethe cognate receptor for the targeting ligand. In this manner, thevector (containing a nucleic acid or a protein) can be selectivelydelivered to a cardiomyocyte cell in, e.g., the myocardium.Methodologies for targeting include conjugates, such as those describedin U.S. Pat. No. 5,391,723 to Priest. Another example of a well-knowntargeting vehicle is a liposome. Liposomes are commercially availablefrom Gibco BRL (Life Technologies Inc., Rockville, Md.). Numerousmethods are published for making targeted liposomes. Preferably, themolecules of the invention are targeted for delivery to cardiomyocytes,and/or vascular endothelial cells.

In general, the vectors useful in the invention include, but are notlimited to, plasmids, phagemids, viruses, other vehicles derived fromviral or bacterial sources that have been manipulated by the insertionor incorporation of the nucleic acid sequences of the invention, andadditional nucleic acid fragments (e.g., enhancers, promoters) which canbe attached to the nucleic acid sequences of the invention. Viralvectors are a preferred type of vector and include, but are not limitedto, nucleic acid sequences from the following viruses: adenovirus;adeno-associated virus; retrovirus, such as Moloney murine leukemiavirus; Harvey murine sarcoma virus; murine mammary tumor virus; rousesarcoma virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses;papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNAviruses such as a retrovirus. One can readily employ other vectors notnamed but known in the art.

A particularly preferred virus for certain applications is theadeno-associated virus, a double-stranded DNA virus. Theadeno-associated virus is capable of infecting a wide range of celltypes and species and can be engineered to be replication-deficienti.e., capable of directing synthesis of the desired proteins, butincapable of manufacturing an infectious particle. It further hasadvantages, such as heat and lipid solvent stability, high transductionfrequencies in cells of diverse lineages, including hematopoietic cells,and lack of superinfection inhibition thus allowing multiple series oftransductions. Reportedly, the adeno-associated virus can integrate intohuman cellular DNA in a site-specific manner, thereby minimizing thepossibility of insertional mutagenesis and variability of inserted geneexpression. In addition, wild-type adeno-associated virus infectionshave been followed in tissue culture for greater than 100 passages inthe absence of selective pressure, implying that the adeno-associatedvirus genomic integration is a relatively stable event. Theadeno-associated virus can also function in an extrachromosomal fashion.

In general, other preferred viral vectors are based on non-cytopathiceukaryotic viruses in which non-essential genes have been replaced withthe gene of interest. Non-cytopathic viruses include retroviruses, thelife cycle of which involves reverse transcription of genomic viral RNAinto DNA with subsequent proviral integration into host cellular DNA.Adenoviruses and retroviruses have been approved for human gene therapytrials. In general, the retroviruses are replication-deficient. Suchgenetically altered retroviral expression vectors have general utilityfor the high-efficiency transduction of genes in vivo. Standardprotocols for producing replication-deficient retroviruses (includingthe steps of incorporation of exogenous genetic material into a plasmid,transfection of a packaging cell line with plasmid, production ofrecombinant retroviruses by the packaging cell line, collection of viralparticles from tissue culture media, and infection of the target cellswith viral particles) are provided in Kriegler, M., “Gene Transfer andExpression, A Laboratory Manual,” W.H. Freeman C.O., New York (1990) andMurry, E. J. Ed. “Methods in Molecular Biology,” vol. 7, Humana Press,Inc., Cliffton, N.J. (1991).

Another preferred retroviral vector is the vector derived from theMoloney murine leukemia virus, as described in Nabel, E. G., et al.,Science, 1990, 249:1285-1288. These vectors reportedly were effectivefor the delivery of genes to all three layers of the arterial wall,including the media. Other preferred vectors are disclosed in Flugelman,et al., Circulation, 1992, 85:1110-1117. Additional vectors that areuseful for delivering molecules of the invention are described in U.S.Pat. No. 5,674,722 by Mulligan, et al.

In addition to the foregoing vectors, other delivery methods may be usedto deliver a molecule of the invention to a cell such as a cardiomyocyteand/or a vascular endothelial cell, and facilitate uptake thereby.

A preferred such delivery method of the invention is a colloidaldispersion system. Colloidal dispersion systems include lipid-basedsystems including oil-in-water emulsions, micelles, mixed micelles, andliposomes. A preferred colloidal system of the invention is a liposome.Liposomes are artificial membrane vessels which are useful as a deliveryvector in vivo or in vitro. It has been shown that large unilamellarvessels (LUV), which range in size from 0.2-4.0 μm can encapsulate largemacromolecules. RNA, DNA, and intact virions can be encapsulated withinthe aqueous interior and be delivered to cells in a biologically activeform (Fraley, et al., Trends Biochem. Sci., 1981, 6:77). In order for aliposome to be an efficient gene transfer vector, one or more of thefollowing characteristics should be present: (1) encapsulation of thegene of interest at high efficiency with retention of biologicalactivity; (2) preferential and substantial binding to a target cell incomparison to non-target cells; (3) delivery of the aqueous contents ofthe vesicle to the target cell cytoplasm at high efficiency; and (4)accurate and effective expression of genetic information.

Liposomes may be targeted to a particular tissue, such as the myocardiumor the vascular cell wall, by coupling the liposome to a specific ligandsuch as a monoclonal antibody, sugar, glycolipid, or protein. Ligandswhich may be useful for targeting a liposome to the vascular wallinclude, but are not limited to, the viral coat protein of theHemagglutinating virus of Japan. Additionally, the vector may be coupledto a nuclear targeting peptide, which will direct the nucleic acid tothe nucleus of the host cell.

Liposomes are commercially available from Gibco BRL, for example, asLIPOFECTIN™ and LIPOFECTACE™, which are formed of cationic lipids suchas N-[1-(2, 3-dioleyloxy)-propyl]-N,N,N-trimethylammonium chloride(DOTMA) and dimethyl dioctadecylammonium bromide (DDAB). Methods formaking liposomes are well known in the art and have been described inmany publications. Liposomes also have been reviewed by Gregoriadis, G.in Trends in Biotechnology, V. 3, p. 235-241 (1985). Novel liposomes forthe intracellular delivery of macromolecules, including nucleic acids,are also described in PCT International application no. PCT/US96/07572(Publication No. WO 96/40060, entitled “Intracellular Delivery ofMacromolecules”).

In one particular embodiment, the preferred vehicle is a biocompatiblemicro particle or implant that is suitable for implantation into themammalian recipient. Exemplary bioerodible implants that are useful inaccordance with this method are described in PCT Internationalapplication no. PCT/US95/03307 (Publication No. WO 95/24929, entitled“Polymeric Gene Delivery System”, which claims priority to U.S. patentapplication Ser. No. 213,668, filed Mar. 15, 1994). PCT/US95/03307describes a biocompatible, preferably biodegradable polymeric matrix forcontaining an exogenous gene under the control of an appropriatepromoter. The polymeric matrix is used to achieve sustained release ofthe exogenous gene in the patient. In accordance with the instantinvention, the nucleic acids described herein are encapsulated ordispersed within the biocompatible, preferably biodegradable polymericmatrix disclosed in PCT/US95/03307. The polymeric matrix preferably isin the form of a micro particle such as a micro sphere (wherein anucleic acid is dispersed throughout a solid polymeric matrix) or amicrocapsule (wherein a nucleic acid is stored in the core of apolymeric shell). Other forms of the polymeric matrix for containing thenucleic acids of the invention include films, coatings, gels, implants,and stents. The size and composition of the polymeric matrix device isselected to result in favorable release kinetics in the tissue intowhich the matrix device is implanted. The size of the polymeric matrixdevice further is selected according to the method of delivery which isto be used, typically injection into a tissue or administration of asuspension by aerosol into the nasal and/or pulmonary areas. Thepolymeric matrix composition can be selected to have both favorabledegradation rates and also to be formed of a material which isbioadhesive, to further increase the effectiveness of transfer when thedevice is administered to a vascular surface. The matrix compositionalso can be selected not to degrade, but rather, to release by diffusionover an extended period of time.

Both non-biodegradable and biodegradable polymeric matrices can be usedto deliver the nucleic acids of the invention to the subject.Biodegradable matrices are preferred. Such polymers may be natural orsynthetic polymers. Synthetic polymers are preferred. The polymer isselected based on the period of time over which release is desired,generally in the order of a few hours to a year or longer. Typically,release over a period ranging from between a few hours and three totwelve months is most desirable. The polymer optionally is in the formof a hydrogel that can absorb up to about 90% of its weight in water andfurther, optionally is cross-linked with multi-valent ions or otherpolymers.

In general, the nucleic acids of the invention are delivered using thebioerodible implant by way of diffusion, or more preferably, bydegradation of the polymeric matrix. Exemplary synthetic polymers whichcan be used to form the biodegradable delivery system include:polyamides, polycarbonates, polyalkylenes, polyalkylene glycols,polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols,polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyglycolides,polysiloxanes, polyurethanes and copolymers thereof, alkyl cellulose,hydroxyalkyl celluloses, cellulose ethers, cellulose esters,nitrocelluloses, polymers of acrylic and methacrylic esters, methylcellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propylmethyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate,cellulose propionate, cellulose acetate butyrate, cellulose acetatephthalate, carboxylethyl cellulose, cellulose triacetate, cellulosesulphate sodium salt, poly(methylmethacrylate), poly(ethylmethacrylate),poly(butylmethacrylate), poly(isobutylmethacrylate),poly(hexylmethacrylate), poly(isodecylmethacrylate),poly(laurylmethacrylate), poly(phenylmethacrylate),poly(methylacrylate), poly(isopropylacrylate), poly(isobutylacrylate),poly(octadecylacrylate), polyethylene, polypropylene,poly(ethyleneglycol), poly(ethyleneoxide), poly(ethyleneterephthalate),poly(vinyl alcohols), polyvinyl acetate, poly vinyl chloride,polystyrene and polyvinylpyrrolidone.

Examples of non-biodegradable polymers include ethylene vinyl acetate,poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof.

Examples of biodegradable polymers include synthetic polymers such aspolymers of lactic acid and glycolic acid, polyanhydrides,poly(ortho)esters, polyurethanes, poly(butic acid), poly(valeric acid),and poly(lactide-cocaprolactone), and natural polymers such as alginateand other polysaccharides including dextran and cellulose, collagen,chemical derivatives thereof (substitutions, additions of chemicalgroups, for example, alkyl, alkylene, hydroxylations, oxidations, andother modifications routinely made by those skilled in the art), albuminand other hydrophilic proteins, zein and other prolamines andhydrophobic proteins, copolymers and mixtures thereof. In general, thesematerials degrade either by enzymatic hydrolysis or exposure to water invivo, by surface or bulk erosion.

Bioadhesive polymers of particular interest include bioerodiblehydrogels described by H. S. Sawhney, C. P. Pathak and J. A. Hubell inMacromolecules, 1993, 26, 581-587, the teachings of which areincorporated herein, polyhyaluronic acids, casein, gelatin, glutin,polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methylmethacrylates), poly(ethyl methacrylates), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), and poly(octadecyl acrylate). Thus, the invention provides acomposition of the above-described molecules of the invention for use asa medicament, methods for preparing the medicament and methods for thesustained release of the medicament in vivo.

Compaction agents also can be used in combination with a vector of theinvention. A “compaction agent”, as used herein, refers to an agent,such as a histone, that neutralizes the negative charges on the nucleicacid and thereby permits compaction of the nucleic acid into a finegranule. Compaction of the nucleic acid facilitates the uptake of thenucleic acid by the target cell. The compaction agents can be usedalone, e.g., to deliver an isolated nucleic acid of the invention in aform that is more efficiently taken up by the cell or, more preferably,in combination with one or more of the above-described vectors.

Other exemplary compositions that can be used to facilitate uptake by atarget cell of the nucleic acids of the invention include calciumphosphate and other chemical mediators of intracellular transport,microinjection compositions, electroporation and homologousrecombination compositions (e.g., for integrating a nucleic acid into apreselected location within the target cell chromosome).

The term “facilitate uptake” of a molecule into a cell according to theinvention has the following meanings depending upon the nature of themolecule. For an isolated nucleic acid it is meant to describe entry ofthe nucleic acid through the cell membrane and into the cell nucleus,where upon the “nucleic acid transgene” can utilize the cell machineryto produce functional polypeptides encoded by the nucleic acid. By“nucleic acid transgene” it is meant to describe all of the nucleicacids of the invention with or without the associated vectors. For apolypeptide, it is meant to describe entry of the polypeptide throughthe cell membrane and into the cell cytoplasm, and if necessary,utilization of the cell cytoplasmic machinery to functionally modify thepolypeptide (e.g., to an active form).

Various techniques may be employed for introducing nucleic acids of theinvention into cells, depending on whether the nucleic acids areintroduced in vitro or in vivo in a host. Such techniques includetransfection of nucleic acid-CaPO₄ precipitates, transfection of nucleicacids associated with DEAE, transfection with a retrovirus including thenucleic acid of interest, liposome mediated transfection, and the like.For certain uses, it is preferred to target the nucleic acid toparticular cells. In such instances, a vehicle used for delivering anucleic acid of the invention into a cell (e.g., a liposome, aretrovirus, or other virus) can have a targeting molecule attachedthereto. For example, a molecule such as an antibody specific for asurface membrane protein on the target cell or a ligand for a receptoron the target cell can be bound to or incorporated within the nucleicacid delivery vehicle. For example, where liposomes are employed todeliver the nucleic acids of the invention, proteins which bind to asurface membrane protein associated with endocytosis may be incorporatedinto the liposome formulation for targeting and/or to facilitate uptake.Such proteins include capsid proteins or fragments thereof tropic for aparticular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half life, and the like.Polymeric delivery systems also have been used successfully to delivernucleic acids into cells, as is known by those skilled in the art. Suchsystems even permit oral delivery of nucleic acids.

The invention also provides methods for the diagnosis and therapy ofvascular and cardiovascular disorders. Such disorders include myocardialinfarction, stroke, arteriosclerosis, heart failure, and cardiachypertrophy.

The methods of the invention are useful in both the acute and theprophylactic treatment of any of the foregoing conditions. As usedherein, an acute treatment refers to the treatment of subjects having aparticular condition. Prophylactic treatment refers to the treatment ofsubjects at risk of having the condition, but not presently having orexperiencing the symptoms of the condition.

In its broadest sense, the terms “treatment” or “to treat” refer to bothacute and prophylactic treatments. If the subject in need of treatmentis experiencing a condition (or has or is having a particularcondition), then treating the condition refers to ameliorating, reducingor eliminating the condition or one or more symptoms arising from thecondition. In some preferred embodiments, treating the condition refersto ameliorating, reducing or eliminating a specific symptom or aspecific subset of symptoms associated with the condition. If thesubject in need of treatment is one who is at risk of having acondition, then treating the subject refers to reducing the risk of thesubject having the condition.

Stroke (also referred to herein as ischemic stroke and/orcerebrovascular ischemia) is often cited as the third most common causeof death in the industrial world, ranking behind ischemic heart diseaseand cancer. Strokes are responsible for about 300,000 deaths annually inthe United States and are a leading cause of hospital admissions andlong-term disabilities. Accordingly, the socioeconomic impact of strokeand its attendant burden on society is practically immeasurable.

“Stroke” is defined by the World Health Organization as a rapidlydeveloping clinical sign of focal or global disturbance of cerebralfunction with symptoms lasting at least 24 hours. Strokes are alsoimplicated in deaths where there is no apparent cause other than aneffect of vascular origin.

Strokes are typically caused by blockages or occlusions of the bloodvessels to the brain or within the brain. With complete occlusion,arrest of cerebral circulation causes cessation of neuronal electricalactivity within seconds. Within a few minutes after the deterioration ofthe energy state and ion homeostasis, depletion of high energyphosphates, membrane ion pump failure, efflux of cellular potassium,influx of sodium chloride and water, and membrane depolarization occur.If the occlusion persists for more than five to ten minutes,irreversible damage results. With incomplete ischemia, however, theoutcome is difficult to evaluate and depends largely on residualperfusion and the availability of oxygen. After a thrombotic occlusionof a cerebral vessel, ischemia is rarely total. Some residual perfusionusually persists in the ischemic area, depending on collateral bloodflow and local perfusion pressure.

Cerebral blood flow can compensate for drops in mean arterial bloodpressure from 90 to 60 mm Hg by autoregulation. This phenomenon involvesdilatation of downstream resistant vessels. Below the lower level ofautoregulation (about 60 mm Hg), vasodilatation is inadequate and thecerebral blood flow falls. The brain, however, has perfusion reservesthat can compensate for the fall in cerebral blood flow. This reserveexists because under normal conditions only about 35% of the oxygendelivered by the blood is extracted. Therefore, increased oxygenextraction can take place, provided that normoxia and normocapnea exist.When distal blood pressure falls below approximately 30 mm Hg, the twocompensatory mechanisms (autoregulation and perfusion reserve) areinadequate to prevent failure of oxygen delivery.

As blood flow drops below the ischemic threshold of 23 ml/100 g/minute,symptoms of tissue hypoxia develop. Severe ischemia may be lethal. Whenthe ischemia is moderate, it will result in “penumbra.” In theneurological context, penumbra refers to a zone of brain tissue withmoderate ischemia and paralyzed neuronal function, which is reversiblewith restoration of adequate perfusion. The penumbra forms a zone ofcollaterally perfused tissue surrounding a core of severe ischemia inwhich an infarct has developed. In other words, the penumbra is thetissue area that can be saved, and is essentially in a state betweenlife and death.

Although an ischemic event can occur anywhere in the vascular system,the carotid artery bifurcation and the origin of the internal carotidartery are the most frequent sites for thrombotic occlusions of cerebralblood vessels, which result in cerebral ischemia. The symptoms ofreduced blood flow due to stenosis or thrombosis are similar to thosecaused by middle cerebral artery disease. Flow through the ophthalmicartery is often affected sufficiently to produce amaurosis fugax ortransient monocular blindness. Severe bilateral internal carotid arterystenosis may result in cerebral hemispheric hypoperfusion. Thismanifests with acute headache ipsilateral to the acutely ischemichemisphere. Occlusions or decrease of the blood flow with resultingischemia of one anterior cerebral artery distal to the anteriorcommunicating artery produces motor and cortical sensory symptoms in thecontralateral leg and, less often, proximal arm. Other manifestations ofocclusions or underperfusion of the anterior cerebral artery includegait ataxia and sometimes urinary incontinence due to damage to theparasagital frontal lobe. Language disturbances manifested as decreasedspontaneous speech may accompany generalized depression of psychomotoractivity.

Most ischemic strokes involve portions or all of the territory of themiddle cerebral artery with emboli from the heart or extracranialcarotid arteries accounting for most cases. Emboli may occlude the mainstem of the middle cerebral artery, but more frequently produce distalocclusion of either the superior or the inferior branch. Occlusions ofthe superior branch cause weakness and sensory loss that are greatest inthe face and arm. Occlusions of the posterior cerebral artery distal toits penetrating branches cause complete contralateral loss of vision.Difficulty in reading (dyslexia) and in performing calculations(dyscalculia) may follow ischemia of the dominant posterior cerebralartery. Proximal occlusion of the posterior cerebral artery causesischemia of the branches penetrating to calamic and limbic structures.The clinical results are hemisensory disturbances that may chronicallychange to intractable pain of the defective side (thalamic pain).

A subject having a stroke is so diagnosed by symptoms experienced and/orby a physical examination including interventional andnon-interventional diagnostic tools such as CT and MR imaging. Themethods of the invention are advantageous for the treatment of variousclinical presentations of stroke subjects. A subject having a stroke maypresent with one or more of the following symptoms: paralysis, weakness,decreased sensation and/or vision, numbness, tingling, aphasia (e.g.,inability to speak or slurred speech, difficulty reading or writing),agnosia (i.e., inability to recognize or identify sensory stimuli), lossof memory, co-ordination difficulties, lethargy, sleepiness orunconsciousness, lack of bladder or bowel control and cognitive decline(e.g., dementia, limited attention span, inability to concentrate).Using medical imaging techniques, it may be possible to identify asubject having a stroke as one having an infarct or one havinghemorrhage in the brain.

An important embodiment of the invention is treatment of a subject withan abnormally elevated risk of an ischemic stroke. As used herein,subjects having an abnormally elevated risk of an ischemic stroke are acategory determined according to conventional medical practice (seeearlier discussion); such subjects may also be identified inconventional medical practice as having known risk factors for stroke orhaving increased risk of cerebrovascular events. This category includes,for example, subjects which are having elective vascular surgery.Typically, the risk factors associated with cardiac disease are the sameas are associated with stroke. The primary risk factors includehypertension, hypercholesterolemia, and smoking. Atrial fibrillation orrecent myocardial infarction are also important risk factors. Inaddition, modified levels of expression of a IL1RL-1 nucleic acidmolecule, or an expression product thereof, are also, according to thepresent invention, important risk factors.

As used herein, subjects having an abnormally elevated risk of anischemic stroke also include subjects undergoing surgical or diagnosticprocedures which risk release of emboli, lowering of blood pressure ordecrease in blood flow to the brain, such as carotid endarterectomy,brain angiography, neurosurgical procedures in which blood vessels arecompressed or occluded, cardiac catheterization, angioplasty, includingballoon angioplasty, coronary by-pass surgery, or similar procedures.Subjects having an abnormally elevated risk of an ischemic stroke alsoinclude subjects having any cardiac condition that may lead to decreasedblood flow to the brain, such as atrial fibrillation, ventricaltachycardia, dilated cardiomyopathy and other cardiac conditionsrequiring anticoagulation. Subjects having an abnormally elevated riskof an ischemic stroke also include subjects having conditions includingarteriopathy or brain vasculitis, such as that caused by lupus,congenital diseases of blood vessels, such as CADASIL syndrome, ormigraine, especially prolonged episodes.

The treatment of stroke can be for patients who have experienced astroke or can be a prophylactic treatment. Short term prophylactictreatment is indicated for subjects having surgical or diagnosticprocedures which risk release of emboli, lowering of blood pressure ordecrease in blood flow to the brain, to reduce the injury due to anyischemic event that occurs as a consequence of the procedure. Longerterm or chronic prophylactic treatment is indicated for subjects havingcardiac conditions that may lead to decreased blood flow to the brain,or conditions directly affecting brain vasculature. If prophylactic,then the treatment is for subjects having an abnormally elevated risk ofan ischemic stroke, as described above. If the subject has experienced astroke, then the treatment can include acute treatment. Acute treatmentfor stroke subjects means administration of an agent of the invention atthe onset of symptoms of the condition or within 48 hours of the onset,preferably within 24 hours, more preferably within 12 hours, morepreferably within 6 hours, and even more preferably within 3 hours ofthe onset of symptoms of the condition.

Criteria for defining hypercholesterolemic and/or hypertriglyceridemicsubjects are well known in the art (see, e.g., “Harrison's”).Hypercholesterolemic subjects and hypertriglyceridemic subjects areassociated with increased incidence of premature coronary heart disease.A hypercholesterolemic subject has an LDL level of >160 mg/dL or >130mg/dL and at least two risk factors selected from the group consistingof male gender, family history of premature coronary heart disease,cigarette smoking (more than 10 per day), hypertension, low HDL (<35mg/dL), diabetes mellitus, hyperinsulinemia, abdominal obesity, highlipoprotein (a), and personal history of cerebrovascular disease orocclusive peripheral vascular disease. A hypertriglyceridemic subjecthas a triglyceride (TG) level of >250 mg/dL. Thus, a hyperlipidemicsubject is defined as one whose cholesterol and triglyceride levelsequal or exceed the limits set as described above for both thehypercholesterolemic and hypertriglyceridemic subjects.

“Myocardial infarction” is a focus of necrosis resulting from inadequateperfusion of the cardiac tissue. Myocardial infarction generally occurswith the abrupt decrease in coronary blood flow that follows athrombotic occlusion of a coronary artery previously narrowed byatherosclerosis. Generally, infarction occurs when an atheroscleroticplaque fissures, ruptures, or ulcerates, and a mural thrombus formsleading to coronary artery occlusion.

The diagnosis of myocardial infarction in a subject determines the needfor treating the subject according to the methods of the invention. Anumber of laboratory tests, well known in the art, are described, forexample, in Harrison's. Generally, the tests may be divided into fourmain categories: (1) nonspecific indexes of tissue necrosis andinflammation, (2) electrocardiograms, (3) serum enzyme changes (e.g.,creatine phosphokinase levels), and (4) cardiac imaging. A person ofordinary skill in the art could easily apply any of the foregoing teststo determine when a subject is at risk, is suffering, or has suffered, amyocardial infarction. In addition, increased levels of expression of aIL1RL-1 nucleic acid molecule, or an expression product thereof, arealso, according to the present invention, important risk factors. Apositively identified subject would thus benefit from a method oftreatment of the invention.

According to the invention, the method involves administering to asubject having a myocardial infarction any of the foregoing IL1RL-1molecules in an amount effective to treat the cardiovascular disorder inthe subject. By “having a myocardial infarction” it is meant that thesubject is at risk of developing, is currently having, or has suffered amyocardial infarction. It is believed that immediate administration ofthe molecule would greatly benefit the subject by inhibiting apoptoticcell-death of cardiomyocytes (the cells mostly affected by the infarct)prior to, or following the infarct. By “immediate” it is meant thatadministration occurs before (if it is diagnosed in time), or within 48hours from the myocardial infarct, although administration up to 14 daysafter the episode may also be beneficial to the subject.

Another important embodiment of the invention is the treatment ofischemic injury resulting from arteriosclerosis. Arteriosclerosis is aterm used to describe a thickening and hardening of the arterial wall.It is believed to be responsible for the majority of deaths in theUnited States and in most westernized societies. Atherosclerosis is onetype of arteriosclerosis that is believed to be the cause of mostcoronary artery disease, aortic aneurysm and arterial disease of thelower extremities (including peripheral vascular arteriopathy), as wellas contributing to cerebrovascular disease. Atherosclerosis is theleading cause of death in the United States.

A normal artery typically is lined on its inner-side only by a singlelayer of endothelial cells, the intima. The intima overlays the media,which contains only a single cell type, the smooth muscle cell. Theouter-most layer of the artery is the adventitia. With aging, there is acontinuous increase in the thickness of the intima, believed to resultin part from migration and proliferation of smooth muscle cells from themedia. A similar increase in the thickness of the intima also occurs asa result of various traumatic events or interventions, such as occurswhen, for example, a balloon dilatation procedure causes injury to thevessel wall. The invention is used in connection with treating ischemicinjury resulting from arteriosclerotic conditions. An arterioscleroticcondition as used herein means classical atherosclerosis, acceleratedatherosclerosis, atherosclerosis lesions and any other arterioscleroticconditions characterized by undesirable endothelial and/or vascularsmooth muscle cell proliferation, including vascular complications ofdiabetes.

Another important embodiment of the invention is the treatment of heartfailure. Heart failure is a clinical syndrome of diverse etiologieslinked by the common denominator of impaired heart pumping and ischaracterized by the failure of the heart to pump blood commensuratewith the requirements of the metabolizing tissues, or to do so only froman elevating filling pressure.

Another important embodiment of the invention is the treatment ofcardiac hypertrophy. This condition is typically characterized by leftventricular hypertrophy, usually of a nondilated chamber, withoutobvious antecedent cause. Current methods of diagnosis include theelectrocardiogram and the echocardiogram. Many patients, however, areasymptomatic and may be relatives of patients with known disease.Unfortunately, the first manifestation of the disease may be suddendeath, frequently occurring in children and young adults, often duringor after physical exertion.

Agents for reducing the risk of or treating a cardiovascular disorderinclude those selected from the group consisting of anti-inflammatoryagents, anti-thrombotic agents, anti-platelet agents, fibrinolyticagents, lipid reducing agents, direct thrombin inhibitors, glycoproteinIIb/IIIa receptor inhibitors, agents that bind to cellular adhesionmolecules and inhibit the ability of white blood cells to attach to suchmolecules (e.g. anti-cellular adhesion molecule antibodies), calciumchannel blockers, beta-adrenergic receptor blockers, cyclooxygenase-2inhibitors, angiotensin system inhibitors, and/or any combinationsthereof. One preferred agent is aspirin.

The mode of administration and dosage of a therapeutic agent of theinvention will vary with the particular stage of the condition beingtreated, the age and physical condition of the subject being treated,the duration of the treatment, the nature of the concurrent therapy (ifany), the specific route of administration, and the like factors withinthe knowledge and expertise of the health practitioner.

As described herein, the agents of the invention are administered ineffective amounts to treat any of the foregoing cardiovasculardisorders. In general, an effective amount is any amount that can causea beneficial change in a desired tissue of a subject. Preferably, aneffective amount is that amount sufficient to cause a favorablephenotypic change in a particular condition such as a lessening,alleviation or elimination of a symptom or of a condition as a whole.

In general, an effective amount is that amount of a pharmaceuticalpreparation that alone, or together with further doses, produces thedesired response. This may involve only slowing the progression of thecondition temporarily, although more preferably, it involves halting theprogression of the condition permanently or delaying the onset of orpreventing the condition from occurring. This can be monitored byroutine methods. Generally, doses of active compounds would be fromabout 0.01 mg/kg per day to 1000 mg/kg per day. It is expected thatdoses ranging from 50-500 mg/kg will be suitable, preferably orally andin one or several administrations per day.

Such amounts will depend, of course, on the particular condition beingtreated, the severity of the condition, the subject patient parametersincluding age, physical condition, size and weight, the duration of thetreatment, the nature of concurrent therapy (if any), the specific routeof administration and like factors within the knowledge and expertise ofthe health practitioner. Lower doses will result from certain forms ofadministration, such as intravenous administration. In the event that aresponse in a subject is insufficient at the initial doses applied,higher doses (or effectively higher doses by a different, more localizeddelivery route) may be employed to the extent that patient tolerancepermits. Multiple doses per day are contemplated to achieve appropriatesystemic levels of compounds. It is preferred generally that a maximumdose be used, that is, the highest safe dose according to sound medicaljudgment. It will be understood by those of ordinary skill in the art,however, that a patient may insist upon a lower dose or tolerable dosefor medical reasons, psychological reasons or for virtually any otherreasons.

The agents of the invention may be combined, optionally, with apharmaceutically-acceptable carrier to form a pharmaceuticalpreparation. The term “pharmaceutically-acceptable carrier,” as usedherein, means one or more compatible solid or liquid fillers, diluentsor encapsulating substances which are suitable for administration into ahuman. The term “carrier” denotes an organic or inorganic ingredient,natural or synthetic, with which the active ingredient is combined tofacilitate the application. The components of the pharmaceuticalcompositions also are capable of being co-mingled with the molecules ofthe present invention, and with each other, in a manner such that thereis no interaction which would substantially impair the desiredpharmaceutical efficacy. In some aspects, the pharmaceuticalpreparations comprise an agent of the invention in an amount effectiveto treat a disorder.

The pharmaceutical preparations may contain suitable buffering agents,including: acetic acid in a salt; citric acid in a salt; boric acid in asalt; or phosphoric acid in a salt. The pharmaceutical compositions alsomay contain, optionally, suitable preservatives, such as: benzalkoniumchloride; chlorobutanol; parabens or thimerosal.

A variety of administration routes are available. The particular modeselected will depend, of course, upon the particular drug selected, theseverity of the condition being treated and the dosage required fortherapeutic efficacy. The methods of the invention, generally speaking,may be practiced using any mode of administration that is medicallyacceptable, meaning any mode that produces effective levels of theactive compounds without causing clinically unacceptable adverseeffects. Such modes of administration include oral, rectal, topical,nasal, intradermal, transdermal, or parenteral routes. The term“parenteral” includes subcutaneous, intravenous, intramuscular, orinfusion. Intravenous or intramuscular routes are not particularlysuitable for long-term therapy and prophylaxis. As an example,pharmaceutical compositions for the acute treatment of subjects having amigraine headache may be formulated in a variety of different ways andfor a variety of administration modes including tablets, capsules,powders, suppositories, injections and nasal sprays.

The pharmaceutical preparations may conveniently be presented in unitdosage form and may be prepared by any of the methods well-known in theart of pharmacy. All methods include the step of bringing the activeagent into association with a carrier which constitutes one or moreaccessory ingredients. In general, the compositions are prepared byuniformly and intimately bringing the active compound into associationwith a liquid carrier, a finely divided solid carrier, or both, andthen, if necessary, shaping the product.

Compositions suitable for oral administration may be presented asdiscrete units, such as capsules, tablets, lozenges, each containing apredetermined amount of the active compound. Other compositions includesuspensions in aqueous liquids or non-aqueous liquids such as a syrup,elixir or an emulsion.

Compositions suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of an agent of the invention,which is preferably isotonic with the blood of the recipient. Thisaqueous preparation may be formulated according to known methods usingsuitable dispersing or wetting agents and suspending agents. The sterileinjectable preparation also may be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example, as a solution in 1,3-butane diol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono ordi-glycerides. In addition, fatty acids such as oleic acid may be usedin the preparation of injectables. Formulations suitable for oral,subcutaneous, intravenous, intramuscular, etc. administrations can befound in Remington's Pharmaceutical Sciences, Mack Publishing Co.,Easton, Pa.

Other delivery systems can include time release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of an agent of the present invention, increasingconvenience to the subject and the physician. Many types of releasedelivery systems are available and known to those of ordinary skill inthe art. They include polymer base systems such aspoly(lactide-glycolide), copolyoxalates, polycaprolactones,polyesteramides, polyorthoesters, polyhydroxybutyric acid, andpolyanhydrides. Microcapsules of the foregoing polymers containing drugsare described in, for example, U.S. Pat. No. 5,075,109. Delivery systemsalso include non-polymer systems that are: lipids including sterols suchas cholesterol, cholesterol esters and fatty acids or neutral fats suchas mono-, di-, and tri-glycerides; hydrogel release systems; sylasticsystems; peptide based systems; wax coatings; compressed tablets usingconventional binders and excipients; partially fused implants; and thelike. Specific examples include, but are not limited to: (a) erosionalsystems in which an agent of the invention is contained in a form withina matrix such as those described in U.S. Pat. Nos. 4,452,775; 4,675,189;and 5,736,152; and (b) diffusional systems in which an active componentpermeates at a controlled rate from a polymer such as described in U.S.Pat. Nos. 3,854,480; 5,133,974: and 5,407,686. In addition, pump-basedhardware delivery systems can be used, some of which are adapted forimplantation.

Use of a long-term sustained release implant may be desirable. Long-termrelease, as used herein, means that the implant is constructed andarranged to deliver therapeutic levels of the active ingredient for atleast 30 days, and preferably 60 days. Long-term sustained releaseimplants are well-known to those of ordinary skill in the art andinclude some of the release systems described above. Specific examplesinclude, but are not limited to, long-term sustained release implantsdescribed in U.S. Pat. No. 4,748,024, and Canadian Patent No. 1330939.

The invention also involves the administration, and in some embodimentsco-administration, of agents other than the IL1RL-1 molecules of theinvention (nucleic acids and polypeptides, and/or fragments thereof)that when administered in effective amounts can act cooperatively,additively or synergistically with a molecule of the invention to: (i)modulate cardiac cell anti-apoptotic activity, and (ii) treat any of theconditions in which cardiac cell anti-apoptotic activity of a moleculeof the invention is involved. Agents other than the molecules of theinvention include anti-inflammatory agents, anti-thrombotic agents,anti-coagulants, anti-platelet agents, fibrinolytic agents, lipidreducing agents, direct thrombin inhibitors, glycoprotein IIb/IIIareceptor inhibitors, agents that bind to cellular adhesion molecules andinhibit the ability of white blood cells to attach to such molecules,calcium channel blockers, beta-adrenergic receptor blockers,cyclooxygenase-2 inhibitors, angiotensin system inhibitors,anti-hypertensive agents, and/or combinations thereof.

“Anti-inflammatory” agents include Alclofenac; AlclometasoneDipropionate; Algestone Acetonide; Alpha Amylase; Amcinafal; Amcinafide;Amfenac Sodium; Amiprilose Hydrochloride; Anakinra; Anirolac;Anitrazafen; Apazone; Balsalazide Disodium; Bendazac; Benoxaprofen;Benzydamine Hydrochloride; Bromelains; Broperamole; Budesonide;Carprofen; Cicloprofen; Cintazone; Cliprofen; Clobetasol Propionate;Clobetasone Butyrate; Clopirac; Cloticasone Propionate; CormethasoneAcetate; Cortodoxone; Deflazacort; Desonide; Desoximetasone;Dexamethasone Dipropionate; Diclofenac Potassium; Diclofenac Sodium;Diflorasone Diacetate; Diflumidone Sodium; Diflunisal; Difluprednate;Diftalone; Dimethyl Sulfoxide; Drocinonide; Endrysone; Enlimomab;Enolicam Sodium; Epirizole; Etodolac; Etofenamate; Felbinac; Fenamole;Fenbufen; Fenclofenac; Fenclorac; Fendosal; Fenpipalone; Fentiazac;Flazalone; Fluazacort; Flufenamic Acid; Flumizole; Flunisolide Acetate;Flunixin; Flunixin Meglumine; Fluocortin Butyl; Fluorometholone Acetate;Fluquazone; Flurbiprofen; Fluretofen; Fluticasone Propionate;Furaprofen; Furobufen; Halcinonide; Halobetasol Propionate; HalopredoneAcetate; Ibufenac; Ibuprofen; Ibuprofen Aluminum; Ibuprofen Piconol;Ilonidap; Indomethacin; Indomethacin Sodium; Indoprofen; Indoxole;Intrazole; Isoflupredone Acetate; Isoxepac; Isoxicam; Ketoprofen;Lofemizole Hydrochloride; Lornoxicam; Loteprednol Etabonate;Meclofenamate Sodium; Meclofenamic Acid; Meclorisone Dibutyrate;Mefenamic Acid; Mesalamine; Meseclazone; Methylprednisolone Suleptanate;Morniflumate; Nabumetone; Naproxen; Naproxen Sodium; Naproxol; Nimazone;Olsalazine Sodium; Orgotein; Orpanoxin; Oxaprozin; Oxyphenbutazone;Paranyline Hydrochloride; Pentosan Polysulfate Sodium; PhenbutazoneSodium Glycerate; Pirfenidone; Piroxicam; Piroxicam Cinnamate; PiroxicamOlamine; Pirprofen; Prednazate; Prifelone; Prodolic Acid; Proquazone;Proxazole; Proxazole Citrate; Rimexolone; Romazarit; Salcolex;Salnacedin; Salsalate; Salycilates; Sanguinarium Chloride; Seclazone;Sermetacin; Sudoxicam; Sulindac; Suprofen; Talmetacin; Talniflumate;Talosalate; Tebufelone; Tenidap; Tenidap Sodium; Tenoxicam; Tesicam;Tesimide; Tetrydamine; Tiopinac; Tixocortol Pivalate; Tolmetin; TolmetinSodium; Triclonide; Triflumidate; Zidometacin; Glucocorticoids; andZomepirac Sodium. One preferred anti-inflammatory agent is aspirin.

“Anti-thrombotic” and/or “fibrinolytic” agents include plasminogen (toplasmin via interactions of prekallikrein, kininogens, Factors XII,XIIIa, plasminogen proactivator, and tissue plasminogen activator[TPA])Streptokinase; Urokinase: Anisoylated Plasminogen-StreptokinaseActivator Complex; Pro-Urokinase; (Pro-UK); rTPA (alteplase or activase;“r” denotes recombinant); rPro-UK; Abbokinase; Eminase; SreptaseAnagrelide Hydrochloride; Bivalirudin; Dalteparin Sodium; DanaparoidSodium; Dazoxiben Hydrochloride; Efegatran Sulfate; Enoxaparin Sodium;Ifetroban; Ifetroban Sodium; Tinzaparin Sodium; Retaplase; Trifenagrel;Warfarin; and Dextrans.

“Anti-platelet” agents include Clopridogrel; Sulfinpyrazone; Aspirin;Dipyridamole; Clofibrate; Pyridinol Carbamate; PGE; Glucagon;Antiserotonin drugs; Caffeine; Theophyllin Pentoxifyllin; Ticlopidine;and Anagrelide.

“Lipid reducing” agents include gemfibrozil, cholystyramine, colestipol,nicotinic acid, probucol lovastatin, fluvastatin, simvastatin,atorvastatin, pravastatin, and cirivastatin.

“Direct thrombin inhibitors” include hirudin, hirugen, hirulog,agatroban, PPACK, and thrombin aptamers.

“Glycoprotein IIb/IIIa receptor inhibitors” embraces both antibodies andnon-antibodies, and include, but are not limited, to ReoPro (abcixamab),lamifiban, and tirofiban.

“Calcium channel blockers” are a chemically diverse class of compoundshaving important therapeutic value in the control of a variety ofdiseases including several cardiovascular disorders, such ashypertension, angina, and cardiac arrhythmias (Fleckenstein, Cir. Res.v. 52, (suppl. 1), p. 13-16 (1983); Fleckenstein, Experimental Facts andTherapeutic Prospects, John Wiley, New York (1983); McCall, D., CurrPract Cardiol, v. 10, p. 1-11 (1985)). Calcium channel blockers are aheterogeneous group of drugs that prevent or slow the entry of calciuminto cells by regulating cellular calcium channels. (Remington, TheScience and Practice of Pharmacy, Nineteenth Edition, Mack PublishingCompany, Eaton, Pa., p. 963 (1995)). Most of the currently availablecalcium channel blockers, and useful according to the present invention,belong to one of three major chemical groups of drugs, thedihydropyridines, such as nifedipine, the phenyl alkyl amines, such asverapamil, and the benzothiazepines, such as diltiazem. Other calciumchannel blockers useful according to the invention, include, but are notlimited to, amrinone, amlodipine, bencyclane, felodipine, fendiline,flunarizine, isradipine, nicardipine, nimodipine, perhexilene,gallopamil, tiapamil and tiapamil analogues (such as 1993RO-11-2933),phenytoin, barbiturates, and the peptides dynorphin, omega-conotoxin,and omega-agatoxin, and the like and/or pharmaceutically acceptablesalts thereof.

“Beta-adrenergic receptor blocking agents” are a class of drugs thatantagonize the cardiovascular effects of catecholamines in anginapectoris, hypertension, and cardiac arrhythmias. Beta-adrenergicreceptor blockers include, but are not limited to, atenolol, acebutolol,alprenolol, befunolol, betaxolol, bunitrolol, carteolol, celiprolol,hedroxalol, indenolol, labetalol, levobunolol, mepindolol, methypranol,metindol, metoprolol, metrizoranolol, oxprenolol, pindolol, propranolol,practolol, practolol, sotalolnadolol, tiprenolol, tomalolol, timolol,bupranolol, penbutolol, trimepranol,2-(3-(1,1-dimethylethyl)-amino-2-hydroxypropoxy)-3-pyridenecarbonitrilHCl,1-butylamino-3-(2,5-dichlorophenoxy)-2-propanol,1-isopropylamino-3-(4-(2-cyclopropylmethoxyethyl)phenoxy)-2-propanol,3-isopropylamino-1-(7-methylindan-4-yloxy)-2-butanol,2-(3-t-butylamino-2-hydroxy-propylthio)-4-(5-carbamoyl-2-thienyl)thiazol,7-(2-hydroxy-3-t-butylaminopropoxy)phthalide. The above-identifiedcompounds can be used as isomeric mixtures, or in their respectivelevorotating or dextrorotating form.

Cyclooxygenase-2 (COX-2) is a recently identified form of acyclooxygenase. “Cyclooxygenase” is an enzyme complex present in mosttissues that produces various prostaglandins and thromboxanes fromarachidonic acid. Non-steroidal, anti-inflammatory drugs exert most oftheir anti-inflammatory, analgesic and antipyretic activity and inhibithormone-induced uterine contractions and certain types of cancer growththrough inhibition of the cyclooxygenase (also known as prostaglandinG/H synthase and/or prostaglandin-endoperoxide synthase). Initially,only one form of cyclooxygenase was known, the “constitutive enzyme” orcyclooxygenase-1 (COX-1). It and was originally identified in bovineseminal vesicles.

Cyclooxygenase-2 (COX-2) has been cloned, sequenced and characterizedinitially from chicken, murine and human sources (see, e.g., U.S. Pat.No. 5,543,297, issued Aug. 6, 1996 to Cromlish et al., and assigned toMerck Frosst Canada, Inc., Kirkland, Calif., entitled: “Humancyclooxygenase-2 cDNA and assays for evaluating cyclooxygenase-2activity”). This enzyme is distinct from COX-1. COX-2 is rapidly andreadily inducible by a number of agents including mitogens, endotoxin,hormones, cytokines and growth factors. As prostaglandins have bothphysiological and pathological roles, the constitutive enzyme, COX-1, isresponsible, in large part, for endogenous basal release ofprostaglandins and hence is important in their physiological functionssuch as the maintenance of gastrointestinal integrity and renal bloodflow. By contrast, it is believed that the inducible form, COX-2, ismainly responsible for the pathological effects of prostaglandins whererapid induction of the enzyme would occur in response to such agents asinflammatory agents, hormones, growth factors, and cytokines. Therefore,it is believed that a selective inhibitor of COX-2 has similaranti-inflammatory, antipyretic and analgesic properties to aconventional non-steroidal anti-inflammatory drug, and in additioninhibits hormone-induced uterine contractions and also has potentialanti-cancer effects, but with reduced side effects. In particular, suchCOX-2 inhibitors are believed to have a reduced potential forgastrointestinal toxicity, a reduced potential for renal side effects, areduced effect on bleeding times and possibly a decreased potential toinduce asthma attacks in aspirin-sensitive asthmatic subjects, and aretherefore useful according to the present invention.

A number of selective “COX-2 inhibitors” are known in the art. Theseinclude, but are not limited to, COX-2 inhibitors described in U.S. Pat.No. 5,474,995 “Phenyl heterocycles as COX-2 inhibitors”; U.S. Pat. No.5,521,213 “Diaryl bicyclic heterocycles as inhibitors ofcyclooxygenase-2”; U.S. Pat. No. 5,536,752 “Phenyl heterocycles as COX-2inhibitors”; U.S. Pat. No. 5,550,142 “Phenyl heterocycles as COX-2inhibitors”; U.S. Pat. No. 5,552,422 “Aryl substituted 5,5 fusedaromatic nitrogen compounds as anti-inflammatory agents”; U.S. Pat. No.5,604,253 “N-Benzylindol-3-yl propanoic acid derivatives ascyclooxygenase inhibitors”; U.S. Pat. No. 5,604,260“5-Methanesulfonamido-1-indanones as an inhibitor of cyclooxygenase-2”;U.S. Pat. No. 5,639,780 N-Benzyl indol-3-yl butanoic acid derivatives ascyclooxygenase inhibitors“; U.S. Pat. No. 5,677,318Diphenyl-1,2-3-thiadiazoles as anti-inflammatory agents”; U.S. Pat. No.5,691,374 “Diaryl-5-oxygenated-2-(5H)-furanones as COX-2 inhibitors”;U.S. Pat. No. 5,698,584 “3,4-Diaryl-2-hydroxy-2,5-dihydrofurans asprodrugs to COX-2 inhibitors”; U.S. Pat. No. 5,710,140 “Phenylheterocycles as COX-2 inhibitors”; U.S. Pat. No. 5,733,909 “Diphenylstilbenes as prodrugs to COX-2 inhibitors”; U.S. Pat. No. 5,789,413“Alkylated styrenes as prodrugs to COX-2 inhibitors”; U.S. Pat. No.5,817,700 “Bisaryl cyclobutenes derivatives as cyclooxygenaseinhibitors”; U.S. Pat. No. 5,849,943 “Stilbene derivatives useful ascyclooxygenase-2 inhibitors”; U.S. Pat. No. 5,861,419 “Substitutedpyridines as selective cyclooxygenase-2 inhibitors”; U.S. Pat. No.5,922,742 “Pyridinyl-2-cyclopenten-1-ones as selective cyclooxygenase-2inhibitors”; U.S. Pat. No. 5,925,631 “Alkylated styrenes as prodrugs toCOX-2 inhibitors”; all of which are commonly assigned to Merck FrosstCanada, Inc. (Kirkland, Calif. or Merck & Co., Inc. (Rahway, N.J.).Additional COX-2 inhibitors are also described in U.S. Pat. No.5,643,933, assigned to G. D. Searle & Co. (Skokie, Ill.), entitled:“Substituted sulfonylphenylheterocycles as cyclooxygenase-2 and5-lipoxygenase inhibitors.”

A number of the above-identified COX-2 inhibitors are prodrugs ofselective COX-2 inhibitors, and exert their action by conversion in vivoto the active and selective COX-2 inhibitors. The active and selectiveCOX-2 inhibitors formed from the above-identified COX-2 inhibitorprodrugs are described in detail in WO 95/00501, published Jan. 5, 1995,WO 95/18799, published Jul. 13, 1995 and U.S. Pat. No. 5,474,995, issuedDec. 12, 1995. Given the teachings of U.S. Pat. No. 5,543,297, entitled:“Human cyclooxygenase-2 cDNA and assays for evaluating cyclooxygenase-2activity,” a person of ordinary skill in the art would be able todetermine whether an agent is a selective COX-2 inhibitor or a precursorof a COX-2 inhibitor, and therefore part of the present invention.

An “angiotensin system inhibitor” is an agent that interferes with thefunction, synthesis or catabolism of angiotensin II. These agentsinclude, but are not limited to, angiotensin-converting enzyme (ACE)inhibitors, angiotensin II antagonists, angiotensin II receptorantagonists, agents that activate the catabolism of angiotensin II, andagents that prevent the synthesis of angiotensin I from whichangiotensin II is ultimately derived. The renin-angiotensin system isinvolved in the regulation of hemodynamics and water and electrolytebalance. Factors that lower blood volume, renal perfusion pressure, orthe concentration of Na⁺ in plasma tend to activate the system, whilefactors that increase these parameters tend to suppress its function.

Angiotensin I and angiotensin II are synthesized by the enzymaticrenin-angiotensin pathway. The synthetic process is initiated when theenzyme renin acts on angiotensinogen, a pseudoglobulin in blood plasma,to produce the decapeptide angiotensin I. Angiotensin I is converted byangiotensin converting enzyme (ACE) to angiotensin II (angiotensin-[1-8]octapeptide). The latter is an active pressor substance which has beenimplicated as a causative agent in several forms of hypertension invarious mammalian species, e.g., humans.

Angiotensin (renin-angiotensin) system inhibitors are compounds that actto interfere with the production of angiotensin II from angiotensinogenor angiotensin I or interfere with the activity of angiotensin II. Suchinhibitors are well known to those of ordinary skill in the art andinclude compounds that act to inhibit the enzymes involved in theultimate production of angiotensin II, including renin and ACE. Theyalso include compounds that interfere with the activity of angiotensinII, once produced. Examples of classes of such compounds includeantibodies (e.g., to renin), amino acids and analogs thereof (includingthose conjugated to larger molecules), peptides (including peptideanalogs of angiotensin and angiotensin I), pro-renin related analogs,etc. Among the most potent and useful renin-angiotensin systeminhibitors are renin inhibitors, ACE inhibitors, and angiotensin IIantagonists. In a preferred embodiment of the invention, therenin-angiotensin system inhibitors are renin inhibitors, ACEinhibitors, and angiotensin II antagonists.

“Angiotensin II antagonists” are compounds which interfere with theactivity of angiotensin II by binding to angiotensin II receptors andinterfering with its activity. Angiotensin II antagonists are well knownand include peptide compounds and non-peptide compounds. Mostangiotensin II antagonists are slightly modified congeners in whichagonist activity is attenuated by replacement of phenylalanine inposition 8 with some other amino acid; stability can be enhanced byother replacements that slow degeneration in vivo. Examples ofangiotensin II antagonists include: peptidic compounds (e.g., saralasin,[(San¹)(Val⁵)(Ala⁸)] angiotensin-(1-8) octapeptide and related analogs);N-substituted imidazole-2-one (U.S. Pat. No. 5,087,634); imidazoleacetate derivatives including 2-N-butyl-4-chloro-1-(2-chlorobenzile),imidazole-5-acetic acid (see Long et al., J. Pharmacol. Exp. Ther.247(1), 1-7 (1988)); 4, 5, 6, 7-tetrahydro-1H-imidazo [4, 5-c]pyridine-6-carboxylic acid and analog derivatives (U.S. Pat. No.4,816,463); N2-tetrazole beta-glucuronide analogs (U.S. Pat. No.5,085,992); substituted pyrroles, pyrazoles, and tryazoles (U.S. Pat.No. 5,081,127); phenol and heterocyclic derivatives such as 1,3-imidazoles (U.S. Pat. No. 5,073,566); imidazo-fused 7-member ringheterocycles (U.S. Pat. No. 5,064,825); peptides (e.g., U.S. Pat. No.4,772,684); antibodies to angiotensin II (e.g., U.S. Pat. No.4,302,386); and aralkyl imidazole compounds such as biphenyl-methylsubstituted imidazoles (e.g., EP Number 253,310, Jan. 20, 1988); ES8891(N-morpholinoacetyl-(-1-naphthyl)-L-alanyl-(4, thiazolyl)-L-alanyl (35,45)-4-amino-3-hydroxy-5-cyclo-hexapentanoyl-N-hexylamide, SankyoCompany, Ltd., Tokyo, Japan); SKF108566(E-alpha-2-[2-butyl-1-(carboxyphenyl)methyl]1H-imidazole-5-yl[methylane]-2-thiophenepropanoic acid, Smith KlineBeecham Pharmaceuticals, PA); Losartan (DUP753/MK954, DuPont MerckPharmaceutical Company); Remikirin (RO42-5892, F. Hoffman LaRoche AG);A₂ agonists (Marion Merrill Dow) and certain non-peptide heterocycles(G.D. Searle and Company).

“Angiotensin converting enzyme,” (ACE), is an enzyme which catalyzes theconversion of angiotensin I to angiotensin II. ACE inhibitors includeamino acids and derivatives thereof, peptides, including di- andtripeptides and antibodies to ACE which intervene in therenin-angiotensin system by inhibiting the activity of ACE therebyreducing or eliminating the formation of pressor substance angiotensinII. ACE inhibitors have been used medically to treat hypertension,congestive heart failure, myocardial infarction and renal disease.Classes of compounds known to be useful as ACE inhibitors includeacylmercapto and mercaptoalkanoyl prolines such as captopril (U.S. Pat.No. 4,105,776) and zofenopril (U.S. Pat. No. 4,316,906), carboxyalkyldipeptides such as enalapril (U.S. Pat. No. 4,374,829), lisinopril (U.S.Pat. No. 4,374,829), quinapril (U.S. Pat. No. 4,344,949), ramipril (U.S.Pat. No. 4,587,258), and perindopril (U.S. Pat. No. 4,508,729),carboxyalkyl dipeptide mimics such as cilazapril (U.S. Pat. No.4,512,924) and benazapril (U.S. Pat. No. 4,410,520), phosphinylalkanoylprolines such as fosinopril (U.S. Pat. No. 4,337,201) and trandolopril.

“Renin inhibitors” are compounds which interfere with the activity ofrenin. Renin inhibitors include amino acids and derivatives thereof,peptides and derivatives thereof, and antibodies to renin. Examples ofrenin inhibitors that are the subject of United States patents are asfollows: urea derivatives of peptides (U.S. Pat. No. 5,116,835); aminoacids connected by nonpeptide bonds (U.S. Pat. No. 5,114,937); di- andtri-peptide derivatives (U.S. Pat. No. 5,106,835); amino acids andderivatives thereof (U.S. Pat. Nos. 5,104,869 and 5,095,119); diolsulfonamides and sulfinyls (U.S. Pat. No. 5,098,924); modified peptides(U.S. Pat. No. 5,095,006); peptidyl beta-aminoacyl aminodiol carbamates(U.S. Pat. No. 5,089,471); pyrolimidazolones (U.S. Pat. No. 5,075,451);fluorine and chlorine statine or statone containing peptides (U.S. Pat.No. 5,066,643); peptidyl amino diols (U.S. Pat. Nos. 5,063,208 and4,845,079); N-morpholino derivatives (U.S. Pat. No. 5,055,466);pepstatin derivatives (U.S. Pat. No. 4,980,283); N-heterocyclic alcohols(U.S. Pat. No. 4,885,292); monoclonal antibodies to renin (U.S. Pat. No.4,780,401); and a variety of other peptides and analogs thereof (U.S.Pat. Nos. 5,071,837, 5,064,965, 5,063,207, 5,036,054, 5,036,053,5,034,512, and 4,894,437).

Agents that bind to cellular adhesion molecules and inhibit the abilityof white blood cells to attach to such molecules include polypeptideagents. Such polypeptides include polyclonal and monoclonal antibodies,prepared according to conventional methodology. Such antibodies alreadyare known in the art and include anti-ICAM 1 antibodies as well as othersuch antibodies described above.

Anticoagulant agents include, but are not limited to, Ancrod;Anticoagulant Citrate Dextrose Solution; Anticoagulant Citrate PhosphateDextrose Adenine Solution; Anticoagulant Citrate Phosphate DextroseSolution; Anticoagulant Heparin Solution; Anticoagulant Sodium CitrateSolution; Ardeparin Sodium; Bivalirudin; Bromindione; Dalteparin Sodium;Desirudin; Dicumarol; Heparin Calcium; Heparin Sodium; Lyapolate Sodium;Nafamostat Mesylate; Phenprocoumon; Tinzaparin Sodium; and WarfarinSodium.

Heparin may stabilize symptoms in evolving stroke, but anticoagulantsare useless (and possibly dangerous) in acute completed stroke, and arecontraindicated in hypertensives because of the increased possibility ofhemorrhage into the brain or other organs. Although the timing iscontroversial, anticoagulants may be started to prevent recurrentcardiogenic emboli. Clot lysing agents, including tissue plasminogenactivator and streptokinase, are being evaluated for the very earlytreatment of acute stroke. Nimodipine has recently been shown to improvesurvival and clinical outcome after ischemic stroke.

Other than aspirin, ticlopidine is another antiplatelet agent that hasbeen shown to be beneficial for stroke treatment. Endarterectomy may beindicated in patients with 70 to 99 percent narrowing of a symptomaticinternal carotid artery. However, most authorities agree that carotidendarterectomy is not indicated in patients with TIAs that are referableto the basilar-vertebral system, in patients with significant deficitsfrom prior strokes, or in patients in whom a stroke is evolving.

HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase is themicrosomal enzyme that catalyzes the rate limiting reaction incholesterol biosynthesis (HMG-CoA6Mevalonate). An HMG-CoA reductaseinhibitor inhibits HMG-CoA reductase, and as a result inhibits thesynthesis of cholesterol. A number of HMG-CoA reductase inhibitors hasbeen used to treat subjects with hypercholesterolemia. More recently,HMG-CoA reductase inhibitors have been shown to be beneficial in thetreatment of stroke (Endres M, et al., Proc Natl Acad Sci USA, 1998,95:8880-5).

HMG-CoA reductase inhibitors useful for co-administration with theagents of the invention include, but are not limited to, simvastatin(U.S. Pat. No. 4,444,784); lovastatin (U.S. Pat. No. 4,231,938);pravastatin sodium (U.S. Pat. No. 4,346,227); fluvastatin (U.S. Pat. No.4,739,073); atorvastatin (U.S. Pat. No. 5,273,995); cerivastatin, andnumerous others described in U.S. Pat. No. 5,622,985; U.S. Pat. No.5,135,935; U.S. Pat. No. 5,356,896; U.S. Pat. No. 4,920,109; U.S. Pat.No. 5,286,895; U.S. Pat. No. 5,262,435; U.S. Pat. No. 5,260,332; U.S.Pat. No. 5,317,031; U.S. Pat. No. 5,283,256; U.S. Pat. No. 5,256,689;U.S. Pat. No. 5,182,298; U.S. Pat. No. 5,369,125; U.S. Pat. No.5,302,604; U.S. Pat. No. 5,166,171; U.S. Pat. No. 5,202,327; U.S. Pat.No. 5,276,021; U.S. Pat. No. 5,196,440; U.S. Pat. No. 5,091,386; U.S.Pat. No. 5,091,378; U.S. Pat. No. 4,904,646; U.S. Pat. No. 5,385,932;U.S. Pat. No. 5,250,435; U.S. Pat. No. 5,132,312; U.S. Pat. No.5,130,306; U.S. Pat. No. 5,116,870; U.S. Pat. No. 5,112,857; U.S. Pat.No. 5,102,911; U.S. Pat. No. 5,098,931; U.S. Pat. No. 5,081,136; U.S.Pat. No. 5,025,000; U.S. Pat. No. 5,021,453; U.S. Pat. No. 5,017,716;U.S. Pat. No. 5,001,144; U.S. Pat. No. 5,001,128; U.S. Pat. No.4,997,837; U.S. Pat. No. 4,996,234; U.S. Pat. No. 4,994,494; U.S. Pat.No. 4,992,429; U.S. Pat. No. 4,970,231; U.S. Pat. No. 4,968,693; U.S.Pat. No. 4,963,538; U.S. Pat. No. 4,957,940; U.S. Pat. No. 4,950,675;U.S. Pat. No. 4,946,864; U.S. Pat. No. 4,946,860; U.S. Pat. No.4,940,800; U.S. Pat. No. 4,940,727; U.S. Pat. No. 4,939,143; U.S. Pat.No. 4,929,620; U.S. Pat. No. 4,923,861; U.S. Pat. No. 4,906,657 U.S.Pat. No. 4,906,624; and U.S. Pat. No. 4,897,402, the disclosures ofwhich patents are incorporated herein by reference.

Nitric oxide (NO) has been recognized as a messenger molecule with manyphysiologic roles, in the cardiovascular, neurologic and immune systems(Griffith, T M et al., J Am Coll Cardiol, 1988, 12:797-806). It mediatesblood vessel relaxation, neurotransmission and pathogen suppression. NOis produced from the guanidino nitrogen of L-arginine by NO Synthase(Moncada, S and Higgs, E A, Eur J Clin Invest, 1991, 21:361-374). Agentsthat upregulate endothelial cell Nitric Oxide Synthase include, but arenot limited to, L-arginine, rho GTPase function inhibitors (seeInternational Application WO 99/47153, the disclosure of which isincorporated herein by reference), and agents that disrupt actincytoskeletal organization (see International Application WO 00/03746,the disclosure of which is incorporated herein by reference).

“Co-administering,” as used herein, refers to administeringsimultaneously two or more compounds of the invention (e.g., a IL1RL-1nucleic acid and/or polypeptide, and an agent known to be beneficial inthe treatment of, for example, a cardiovascular condition, e.g., ananticoagulant-), as an admixture in a single composition, orsequentially, close enough in time so that the compounds may exert anadditive or even synergistic effect, i.e., on reducing cardiomyocytecell-death in a cardiovascular condition.

The invention also embraces solid-phase nucleic acid molecule arrays.The array consists essentially of a set of nucleic acid molecules,expression products thereof, or fragments (of either the nucleic acid orthe polypeptide molecule) thereof, the set including a IL1RL-1 nucleicacid molecule and at least one control nucleic acid molecule fixed to asolid substrate. In preferred embodiments, the set of nucleic acidmolecules comprises a maximum number of 100 different nucleic acidmolecules. In important embodiments, the set of nucleic acid moleculescomprises a maximum number of 10 different nucleic acid molecules.

According to the invention, standard hybridization techniques ofmicroarray technology are utilized to assess patterns of nucleic acidexpression and identify nucleic acid expression. Microarray technology,which is also known by other names including:

DNA chip technology, gene chip technology, and solid-phase nucleic acidarray technology, is well known to those of ordinary skill in the artand is based on, but not limited to, obtaining an array of identifiednucleic acid probes (e.g., molecules described elsewhere herein such asIL1RL-1) on a fixed substrate, labeling target molecules with reportermolecules (e.g., radioactive, chemiluminescent, or fluorescent tags suchas fluorescein, Cye3-dUTP, or Cye5-dUTP), hybridizing target nucleicacids to the probes, and evaluating target-probe hybridization. A probewith a nucleic acid sequence that perfectly matches the target sequencewill, in general, result in detection of a stronger reporter-moleculesignal than will probes with less perfect matches. Many components andtechniques utilized in nucleic acid microarray technology are presentedin Nature Genetics, Vol. 21, January 1999, the entire contents of whichis incorporated by reference herein.

According to the present invention, microarray substrates may includebut are not limited to glass, silica, aluminosilicates, borosilicates,metal oxides such as alumina and nickel oxide, various clays,nitrocellulose, or nylon. In all embodiments a glass substrate ispreferred. According to the invention, probes are selected from thegroup of nucleic acids including, but not limited to: DNA, genomic DNA,cDNA, and oligonucleotides; and may be natural or synthetic.Oligonucleotide probes preferably are 20 to 25-mer oligonucleotides andDNA/cDNA probes preferably are 500 to 5000 bases in length, althoughother lengths may be used. Appropriate probe length may be determined byone of ordinary skill in the art by following art-known procedures. Inone embodiment, preferred probes are sets of two or more of the nucleicacid molecules set forth as SEQ ID NOs: 1 and/or 3. Probes may bepurified to remove contaminants using standard methods known to those ofordinary skill in the art such as gel filtration or precipitation.

In one embodiment, the microarray substrate may be coated with acompound to enhance synthesis of the probe on the substrate. Suchcompounds include, but are not limited to, oligoethylene glycols. Inanother embodiment, coupling agents or groups on the substrate can beused to covalently link the first nucleotide or olignucleotide to thesubstrate. These agents or groups may include, but are not limited to:amino, hydroxy, bromo, and carboxy groups. These reactive groups arepreferably attached to the substrate through a hydrocarbyl radical suchas an alkylene or phenylene divalent radical, one valence positionoccupied by the chain bonding and the remaining attached to the reactivegroups. These hydrocarbyl groups may contain up to about ten carbonatoms, preferably up to about six carbon atoms. Alkylene radicals areusually preferred containing two to four carbon atoms in the principalchain. These and additional details of the process are disclosed, forexample, in U.S. Pat. No. 4,458,066, which is incorporated by referencein its entirety.

In one embodiment, probes are synthesized directly on the substrate in apredetermined grid pattern using methods such as light-directed chemicalsynthesis, photochemical deprotection, or delivery of nucleotideprecursors to the substrate and subsequent probe production.

In another embodiment, the substrate may be coated with a compound toenhance binding of the probe to the substrate. Such compounds include,but are not limited to: polylysine, amino silanes, amino-reactivesilanes (Nature Genetics, Vol. 21, January 1999) or chromium. In thisembodiment, presynthesized probes are applied to the substrate in aprecise, predetermined volume and grid pattern, utilizing acomputer-controlled robot to apply probe to the substrate in acontact-printing manner or in a non-contact manner such as ink jet orpiezo-electric delivery. Probes may be covalently linked to thesubstrate with methods that include, but are not limited to,UV-irradiation and heat.

Targets are nucleic acids selected from the group, including but notlimited to, DNA, genomic DNA, cDNA, RNA, mRNA and may be natural orsynthetic. In all embodiments, nucleic acid molecules from subjectssuspected of developing or having a cardiovascular condition, arepreferred. In certain embodiments of the invention, one or more controlnucleic acid molecules are attached to the substrate. Preferably,control nucleic acid molecules allow determination of factors includingbut not limited to: nucleic acid quality and binding characteristics;reagent quality and effectiveness; hybridization success; and analysisthresholds and success. Control nucleic acids may include, but are notlimited to, expression products of genes such as housekeeping genes orfragments thereof.

To select a set of cardiovascular disease markers, the expression datagenerated by, for example, microarray analysis of gene expression, ispreferably analyzed to determine which genes in different categories ofpatients (each category of patients being a different cardiovasculardisorder), are significantly differentially expressed. The significanceof gene expression can be determined using Permax computer software,although any standard statistical package that can discriminatesignificant differences is expression may be used. Permax performspermutation 2-sample t-tests on large arrays of data. For highdimensional vectors of observations, the Permax software computest-statistics for each attribute, and assesses significance using thepermutation distribution of the maximum and minimum overall attributes.The main use is to determine the attributes (genes) that are the mostdifferent between two groups (e.g., control healthy subject and asubject with a particular cardiovascular disorder), measuring “mostdifferent” using the value of the t-statistics, and their significancelevels.

Expression of cardiovascular disease nucleic acid molecules can also bedetermined using protein measurement methods to determine expression ofSEQ ID NOs: 2 and/or 4, e.g., by determining the expression ofpolypeptides encoded by SEQ ID NOs: 1 and/or 3, respectively. Preferredmethods of specifically and quantitatively measuring proteins include,but are not limited to: mass spectroscopy-based methods such as surfaceenhanced laser desorption ionization (SELDI; e.g., Ciphergen ProteinChipSystem), non-mass spectroscopy-based methods, andimmunohistochemistry-based methods such as 2-dimensional gelelectrophoresis.

SELDI methodology may, through procedures known to those of ordinaryskill in the art, be used to vaporize microscopic amounts of tumorprotein and to create a “fingerprint” of individual proteins, therebyallowing simultaneous measurement of the abundance of many proteins in asingle sample. Preferably SELDI-based assays may be utilized tocharacterize cardiovascular conditions as well as stages of suchconditions. Such assays preferably include, but are not limited to thefollowing examples. Gene products discovered by RNA microarrays may beselectively measured by specific (antibody mediated) capture to theSELDI protein disc (e.g., selective SELDI). Gene products discovered byprotein screening (e.g., with 2-D gels), may be resolved by “totalprotein SELDI” optimized to visualize those particular markers ofinterest from among SEQ ID NOs: 1 and/or 3. Predictive models of tumorclassification from SELDI measurement of multiple markers from among SEQID NOs: 1 and/or 3, may be utilized for the SELDI strategies.

The use of any of the foregoing microarray methods to determineexpression of cardiovascular disease nucleic acids can be done withroutine methods known to those of ordinary skill in the art and theexpression determined by protein measurement methods may be correlatedto predetermined levels of a marker used as a prognostic method forselecting treatment strategies for cardiovascular disease patients.

The invention will be more fully understood by reference to thefollowing examples. These examples, however, are merely intended toillustrate the embodiments of the invention and are not to be construedto limit the scope of the invention.

EXAMPLES Example 1 Experimental Protocols: Materials and Methods

Mechanical Strain Device

Experiments of mechanically overloading cardiomyocytes have generallybeen performed by stretching cells with no control of the cardiac cycle,an approach that does not allow distinction between mechanical overloadin contraction versus relaxation. In the present study, we designed andconstructed a unique experimental system that allows preciselycontrolled mechanical strains as well as electrical pacing in culturedcardiomyocytes, to investigate, inter alia, how cardiomyocytemechanotransduction is regulated by the cardiac cycle, and identifygenes that are involved in such regulation.

The Pacing-Strain Device.

The approach to mechanical stimulation used an apparatus that hasmultiple platens that contact the underside of silicone elastomermembranes to apply a spatially isotropic biaxial strain profile to themembrane (Schaffer J L, et al., J Orthop Res, 1993, 12:709-719; and U.S.Provisional patent application filed on Jul. 16, 1999 entitled “ANAPPARATUS FOR STUDYING MYOCARDIAL MECHANICAL OVERLOAD HYPERTROPHY ANDUSES THEREFOR, by Richard T. Lee, and bearing Attorney Docket no.100038.130 and express mail no. EL110243781US). Six individual 78 mmmembranes can be stretched at once with varying amplitudes of strain bycontrolling displacement of each platen with a stepper motor. MeasuredGreen strains are accurate to ˜±0.25% at strains from 1-14% (Cheng G C,et al., Circ Res, 1997, 80:28-36; Brown T D, J Biomechanics, 2000,33:3-14). Throughout this study, 8% biaxial strain was used.

To control the timing of mechanical strain relative to the cardiaccycle, the computer paced each dish electrically, and controlled: thephase between the mechanical strain and the electrical impulse, theelectrical impulse duration, and the voltage of the impulse. Inaddition, the electrical impulses had alternating polarity to minimizeelectrochemical effects such as pH gradients at the electrodes. The twooutputs were each connected to a single set of electrodes in each dish.The dishes were paced in parallel with a resistance of approximately 500ohms per dish.

The positive and negative voltage sources were provided by two powersupplies (6545A, Hewlett Packard Company, Palo Alto, Calif.). Thecontrol circuit was divided into two parts: a high voltage circuit and alow voltage or digital signal circuit. The high voltage circuit was agate that switched the output based on the input signal. The low voltagecircuit accepted two control signals from the computer and accepted thepulse width from a variable resistor, which controlled both the positiveand negative voltage gates. The low voltage circuit allowed a voltagepulse between 0-120V DC amplitude and 2-37 ms duration. Lights providedcontinuous monitoring of the pulses, and the timing of the circuits andcalibration were validated by oscilloscope.

The electrodes for each dish were two arc-shaped AgCl₂ wire electrodesat the base of the inner surface of the dish, just above the deformablemembrane. The electrodes were pre-made, ethanol-sterilized, and placedinto the dish just prior to each experiment to minimize potentialtoxicity from silver. Using this method no cellular death or detachmentwas observed in 24 hr experiments. Each arc was 120 degrees; weperformed a two dimensional finite element analysis to estimate theuniformity of the potential field with this configuration. Thesecalculations estimate a spatial variation in the potential field of{root mean square}=29%. Thus, this system provides highly uniformbiaxial mechanical strain, with a relatively small variation in thevoltage field.

Mechanical Stimulation Protocols.

We imposed strain only during first third of the cardiac cycle byelectrical stimulation for strain imposed during the “systolic phase”,and only during one third of the cardiac cycle in the relaxation phasefor strain imposed during “diastolic phase,” respectively. Conditionsused in this study were: (1) control; (2) strain, no pacing; (3) pacing,no strain; (4) strain imposed during systolic phase; and (5) strainimposed during diastolic phase.

Neonatal rat ventricular myocytes (NRVM) from 1-day old Sprague-Dawleyrats were isolated by previously described methods (Springhorn J P, andClaycomb W C., Biochem J, 1989; 258:73-78; Arstall M A, et al., J MolCell Cardiol, 1998, 30:1019-25). NRVM were plated on the coated membranedish at a density of 2,000,000 cells/dish in DMEM containing 7% FCS andincubated 24 h. Approximate cell confluence was 85-90%. NRVM were thenmade quiescent by washing with 10 ml of Hanks' balanced salt solution(HBSS, 138 mM NaCl, 5.3 mM KCl, 4.0 mM NaHCO₃, 1.3 mM CaCl₂, 0.5 mMMgCl₂, 0.4 mM MgSO₄, 0.4 mM KH₂PO₄, 0.3 mM Na₂HPO₄, 5.6 mM glucose; LifeTechnologies, Inc., Rockville, Md.) twice and incubating with 26 ml ofDMEM containing 0.2% FCS for 48-72 hours.

In these cell culture conditions, cells beat at 40-60 beats/minute. Atthis rate, we have observed negligible competition when pacing at a rateof 70 beats/minute. We performed trial capture experiments; ninelocations on each dish were sampled. Capture efficiency was similar atall locations, and maximal capture occurred at 60 V and above with 10 msof pulse width. Therefore, a voltage of 70 V with 10 ms of impulseduration at a rate of 1.2 Hz (70 beats/minute) was selected. Under theseconditions we did not observe partial cell detachment.

Transcriptional Profiling.

The DNA microarray experiment was performed with rat neonatal cardiacmyocytes cultured on fibronectin-coated membranes with serum-free mediumfor 48 hours. Cells were deformed with an 8% deformation imposed onlyduring systole for a period of 30 minutes, and RNA was prepared after 6hours of subsequent no strain conditions and no pacing conditions. Thistime point was based upon previous studies demonstrating that the genetenascin (positive control for cardiomyocytes) is induced at this timeperiod. The DNA microarray hybridization experiment was performed usingthe Affymatrix GeneChip RGU34A (Affymetrix, Inc., Santa Clara, Calif.).Data were analyzed using Affymatrix software.

Northern Analyses.

The cDNA clones for differentially expressed genes were obtained by PCRusing the GenBank sequences. Each clone was sequenced from both 5′ and3′ ends to confirm identity. Positive elements in the DNA microarraywere confirmed by Northern blot hybridization analysis in at least threeindependent experiments using three different sources of NRVMs. TotalRNA was isolated by the guanidium thiocyanate and phenol chloroformmethod (Chomcyznski, et al., Anal. Biochem., 1987, 162:156-159). ForNorthern blotting, 15 μg RNA was loaded on a 1.0% agarose-formaldehydegel (2.0 mol/l), transferred to a nylon membrane (Amersham PharmaciaBiotech AB, Piscataway, N.J.), and UV cross-linked with a UVStratalinker (Stratagene, Inc., La Jolla, Calif.). Each probe washybridized with ExpressHyb solution (Clontech Labs., Inc., Palo Alto,Calif.) at 68° C. for 1 hour. The membrane was washed with 2×SSC, 0.05%SDS solution for 30 to 40 minutes and three times at room temperatureand 0.1×SSC, 0.1% SDS solution with continuous shaking at 50° C. for 40minutes. The membrane was exposed to film at −80° C., and radiographswere scanned and analyzed with Optimas 5.0 software (Optimas Co./MediaCybernetics, Silver Springs, Md.). Densitometric units were normalizedto the ethidium-stained 28S ribosomal subunit on the membrane.

Results.

FIG. 1 shows the timecourse (early, left; late, right) of the inductionof IL1RL-1 mRNA expression by 8% cyclic mechanical strain in neonatalcardiac myocytes in culture. Maximal induction occurs at 3 hours and issustained for 15 hours.

FIG. 2 shows the effects of 8% mechanical strain, angiotensin receptorblockade (ARB, CP-19116, 100 nM), angiotensin II (Ang II, 50 nM),interleukin-1β(IL-1β, 10 ng/ml), and phorbol ester (PMA, 200 nM) for 3hours on the induction of IL1RL-1 mRNA expression in cultured neonatalrat cardiac myocytes. The induction of IL1RL-1 mRNA expression by strainwas not blocked by angiotensin receptor blockade; furthermore, treatmentwith angiotensin II did not induce IL1RL-1 mRNA expression. Treatmentwith both IL-1β and PMA were associated with an induction of IL1RL-1mRNA expression in the absence of mechanical strain.

FIG. 3 shows the effects of 8% mechanical strain, hydrogen peroxide(H₂O₂, 100 uM) and the antioxidant, TIRON (10 mM) on the induction ofIL1RL-1 mRNA expression. Unlike the mRNA expression of the mechanicallyinduced Tenascin-C gene which is induced by H₂O₂ in the absence ofmechanical strain and blocked by TIRON, H₂O₂ does not induce IL1RL-1 inthe absence of strain and blocks the strain-induced induction ofIL1RL-1. TIRON slightly attenuated the mRNA expression of IL1RL-1 in theabsence and presence of strain.

FIG. 4 shows the effects of actinomycin D (5 μg/ml, left) andcyclohexamide (10 μg/ml, right) on the induction of IL1RL-1 mRNAexpression by 8% mechanical strain. Actinomycin D and cyclohexamide wereapplied during mechanical strain. Actinomycin D blocked the induction ofIL1RL-1 mRNA expression at both 2 and 4 hours suggesting that theinduction of IL1RL-1 in response to strain is due to increasedtranscription of IL1RL-1. The protein synthesis inhibitor, cyclohexamideblocked the induction of IL1RL-1 mRNA expression in response to strainsuggesting that new protein synthesis is required for the induction ofIL1RL-1 mRNA expression.

FIG. 5 shows the effects of 8% mechanical strain alone and incombination with interleukin-1β (IL-1β, 10 ng/ml), and phorbol ester inthe absence of strain (PMA, 100 ng/ml) on IL1RL-1 mRNA expression incultured neonatal cardiac myocytes. Both IL-1β and mechanical strainalone induced IL1RL-1 mRNA expression but the induction of IL1RL-1 bymechanical strain in the presence of IL-1β was not further increasedsuggesting that mechanical strain and IL-1β do not act in a synergisticor additive manner on the induction of IL1RL-1. The strongest inductionof IL1RL-1 mRNA expression is seen with PMA. The rank order potency forthe induction of IL1RL-1 mRNA expression is PMA>strain>IL-1β.

FIG. 6 shows neonatal rat cardiac myocytes were exposed to 8% strain for0, 1, 3, 6, 9 hours. Total RNA was isolated using a RNeasy kit. Five μgof total RNA were size-separated on 1% agarose-formaldehyde gel andtransferred to nylon membrane. After cross-linking with UV light,membrane was hybridized with ³²P-labeled probe specific for V-ATPase Bsubunit. The membrane was then exposed to x-ray film for 3 hours at −80°C. with an intensifying screen.

Example 2 Introduction:

Cytokines and Cardiac Injury.

Stress-activated cytokines participate in many forms of cardiac injuryand pathophysiological conditions, the most characterized ones beingtumor necrosis factor-α, interleukin-1 and interleukin-6. Thesemolecules are not constitutively expressed in the normal heart but arerapidly induced during ischemia and reperfusion or upon hemodynamicoverloading, suggesting that they play an important role in the initialmyocardial response to stress, injury or growth stimuli (Mann D L,Cytokine and Growth Factor Reviews. 1996; 7:341-354; St. John Sutton MG, et al. Circulation. 2000; 101:2981-2988). However, cytokines havealso been shown to be stably expressed in pathologic myocardialconditions including ischemic heart disease and heart failure and areassociated with a poor prognosis (Pulkki K J, et al. Annals of Medicine.1997; 29:339-343; Kubota T, et al Proc Natl Acad Sci. 1998;95:6930-6935; Aukrust P, et al. Am J Cardiol 1999; 83:376-382; MacGowanG A, et al. Am J Cardiol 1997; 79:1128-1132; Roig E, et al. Am J Cardiol1998; 688-690; Tsutamoto T, et al. J Am Coll Cardiol 1998; 31:391-398;Prabhu S D, et al. Circulation. 2000; 101:2103-2109; Murray D R, et al.Annu Rev Immunol. 2000; 18:451-494).

Interleukin-1 signaling through the interleukin-1 receptor is an earlyevent in inflammatory cytokine signaling in many different systems(Trehu E G., Clin Cancer Res. 1996; 8:1341-51). In cardiac injury,interleukin-6 is produced by cardiac myocytes secondary to stimulationwith interleukin-1, tumor necrosis factor-α, or lipopolysaccharide andhas been detected in the post-ischemic lymph during reperfusion ofischemic myocardium (Gwechenberger M, et al. Circulation 1999;99:546-551). Recently recognized is the potential expression ofcounteracting anti-inflammatory cytokines in cardiac disease secondaryto interleukin-1 signaling. Interleukin-4 and interleukin-10 cansuppress the synthesis of tumor necrosis factor-α and enhance therelease of soluble tumor necrosis factor receptors, which are ligandsinks for tumor necrosis factor (Joyce D A., 1994; Eur. J Immunol.11:2699-705). Interleukin-10 is increased in patients with heart failure(Yamaoka M, et al. Jpn Circ J. 1999; 63:951-956) and interleukin-10serum levels are increased when tumor necrosis factor-α serum levels areincreased in patients with dilated cardiomyopathy (Ohtsuka T, et al. JAm Coll Cardiol. 2001; 37:412-417).

T1/ST2 (IL1RL-1): A Novel Mechanically Induced Receptor.

We have identified a novel potential stress-activated signaling pathwayin the heart: regulation of the induction of an interleukin-1 familymember gene, T1/ST2. Little is known of the induction, signaling andfunction of T1/ST2 in any cell type and T1/ST2 was shown in separateareas of investigation to have two seemingly unrelated functions. One ofthese is growth regulation and the other is immune modulation. Bothcompensatory hypertrophic growth and immune/inflammatory modulation areinvolved in the pathophysiology of cardiovascular diseases.

Growth.

The T1/ST2 gene was first identified by its induction following serumstimulation of resting mouse 3T3 fibroblasts, suggesting that the T1/ST2gene participates in growth regulation (Tominaga S., FEBS Letters 1989;258:301-304). The same group later identified a longer transcriptconsisting of transmembrane and cytoplasmic domains homologous to thefull-length interleukin-1 receptor (Yanagisawa K, et al. FEBS Letters.1993; 318:83-87).

Immunity.

T1/ST2 is expressed on T helper-2, but not T helper-1, cells of theadaptive immune system, which produce interleukin-4, interleukin-5 andinterleukin-10 (Yanagisawa K I, et al. J Biochem. 1997; 121:95-103;Coyle A J, et al. J Exp Med. 1999; 190:895-902). T helper-2 cellsmediate beneficial responses to infection, but are detrimental in thedevelopment of allergy and asthma. There is a strong correlation betweenexpression of T1/ST2 and interleukin-4 production on T helper-2 cells(Coyle A J, et al. J Exp Med. 1999; 190:895-902). T1/ST2 plays acritical role in differentiation to and activation of T helper-2 but notT helper-1 cells (O'Neill L A J, et al. Immunology Today. 2000;21:206-209).

Inhibition of T1/ST2 signaling attenuated T helper 2-mediated inductionof eosinophil inflammatory responses in lung and inhibited cytokinesecretion from T helper-2 cells without modifying interferon-gammasecretion from T helper-1 cells (Coyle A J, et al. J Exp Med. 1999;190:895-902). These studies indicate that expression of T1/ST2 can alterthe cytokine profile in favor of expression of interleukin-4,interleukin-5 and interleukin-10. Interleukin-10 has recently been shownto have anti-inflammatory effects in the setting of cardiac injury(Ohtsuka T, et al. J Am Coll Cardiol. 2001; 37:412-417). Similarly, theabsence of T1/ST2 expression could result in a shift towardsinterferon-gamma expression, which may be deleterious followingmyocardial injury.

Taken together, the involvement of T1/ST2 in growth responses and immunefunction coupled with the clinical recognition of the role of cytokinesin the inflammatory response to ischemia/reperfusion are suggestive thatT1/ST2 activation is a growth- or stress-activated signaling pathwaythat contributes to myocardial growth and remodeling.

Phenotype of T1/ST2 Null Mice.

(Townsend M I, et al. J Exp Med. 2000; 191:1069-1075). The absence ofT1/ST2 in T1/ST2 null mice does not compromise their basal immunefunction in the absence of immune challenge. However, T1/ST2 null micehave an impaired ability to generate IL-4, IL-5, and IL-10, but notIFN-γ (a Th1 cytokine) and to generate a T helper-2 inflammatoryresponse during eosinophilic infiltration in the lung (a Th2 response).

We have begun to study the induction of T1/ST2 in cardiac myocytes andits involvement in survival/death signaling within the context of themyocyte signaling pathways. Preliminary studies presented below showthat T1/ST2 is induced in cardiac myocytes in response to interleukin-1and mechanical strain and that the induction of T1/ST2 by interleukin-1may be dependent on NF-κB activation. T1/ST2 mRNA is also induced inhuman adult vascular smooth muscle cells in response to interleukin-1.T1/ST2 protein is expressed in the mouse heart early after myocardialischemia in vivo as well as in human aorta tissue from patients withunstable plaque.

Results: In Vitro Studies.

The following studies demonstrate the induction of T1/ST2 by mechanicalstrain and interleukin-1, possibly through activation of NF-κB. Bothtranscripts of T1/ST2 (that is, IL1RL-1S-soluble- andIL1RL-1M-membrane-) are induced by strain in cardiac myocytes althoughthe more abundant transcript was the soluble isoform. T1/ST2 mRNA isinduced by mechanical strain in cultured neonatal cardiac myocytes (FIG.8).

T1/ST2 mRNA is induced by mechanical strain in cultured neonatal cardiacmyocytes. Neonatal rat ventricular myocytes were isolated by collagenasedigestion, plated on fibronectin-coated silicone membrane dishes at adensity of 3.5 million cells/dish in 13 ml media as previously described(Yamamoto K, et al. J Boil Chem. 1999; 274:21840-21846). This techniqueyields cultures with ≥95% myocytes. Mechanical deformation was appliedusing a device that provides uniform biaxial cyclic strain as previouslydescribed (Yamamoto K, et al. J Biol Chem. 1999; 274:21840-21846). RNAwas extracted (Qiagen) and Northern blotting was performed using as aprobe a ³²P-labelled 600 bp PCR fragment specific to rat T1/ST2. Maximalinduction occurs at 3 hours, is sustained for 9 hours and declines by 15hours.

Both interleukin-1β and mechanical strain each induce T1/ST2 RNA incardiac myocytes (FIG. 9). Shown is the induction of T1/ST2 byinterleukin-1 and strain. We also found that the induction of T1/ST2 bymechanical strain in the presence of interleukin-1β was not furtherincreased suggesting that interleukin-1 does not sensitize myocytes tothe effects of mechanical strain (or vice versa) on the induction ofT1/ST2. The 1 hour time point was included in the event that inductionby strain is saturated at 3 hours and therefore masks an additive effectof interleukin-1β. Shown in the two right lanes are the effects ofphorbol ester (PMA) at 1 and 3 hours. The rank order potency for theinduction of T1/ST2 mRNA expression is PMA>strain>interleukin-1β. Sinceinterleukin-1β signals through NF-κB and PMA through PKC these resultssuggest that NF-κB and PKC activation both participate in the inductionof T1/ST2.

T1/ST2 may be a NF-κB target gene in cardiac myocytes throughinterleukin-1/interleukin-1 receptor signaling (FIG. 10). Previouslyreported by us (Yamamoto K, et al. J Biol Chem. 1999; 274:21840-21846),mechanical strain of cardiac myocytes activates NF-κB. To investigatethe role of NF-κB in interleukin-1β and strain induction of T1/ST2 RNA,we overexpressed IκBα, which decreases NF-κB DNA binding activity.Cultured cardiac myocytes were infected with IκBα overexpressionadenovirus vector or with β-galactosidase control vector and exposed for4 hours to 8% cyclic mechanical strain or interleukin-1 (10 ng/ml). RNAwas analyzed by Northern blotting with ³²P-labeled IL1RL-1 cDNA probe.Ectopic expression of IκBα blocked interleukin-1β induction of T1/ST2-1mRNA and partially blocked strain induction of T1/ST2 mRNA expressionwhen compared with T1/ST2 induction in cells treated with theβ-galactosidase control vector. These results suggest that T1/ST2 is anearly, NF-κB target gene through interleukin-1/interleukin-1 receptorsignaling. In contrast, pathways in addition to NF-κB activation may beinvolved in the induction of T1/ST2 RNA by mechanical strain. T1/ST2mRNA is also induced by interleukin-1 but not PMA or tumor necrosisfactor (TNF) in human adult vascular smooth muscle cells.

In addition to the above-noted results, we have shown that T1/ST2 isinduced secondary to NF-κB activation by interleukin-1 and NF-κB islinked to cardiac myocyte survival. Further in vitro studies areperformed to confirm that T1/ST2 activation is linked to cell growth andsurvival.

In Vivo Studies. Materials and Methods

Experimental Myocardial Infarction in Mice.

Experimental procedures on mice were approved by the Harvard MedicalSchool Standing Committee on Animals. Experimental myocardial infarctionwas created in mice by coronary artery ligation as previously described(13). Hearts were harvested from mice 1 and 3 days after coronary arteryligation followed by perfusion fixation of the heart with Z-Fix (AnatechLTD). Hearts were then immersion fixed in Z-Fix overnight at 4° C. Afterdehydration in graded ethanol solutions, hearts were placed inHisto-Clear (National Diagnostics) and paraffin-embedded. Five microntissue sections were deparaffinized, rehydrated, incubated with 3%hydrogen peroxide, rinsed in water followed by phosphate bufferedsaline. Sections were blocked, incubated in 1:50 anti-mouse ST2 primaryantibody (Morwell Diagnostics) and 1:100 anti-rat HRP conjugatedsecondary antibody (Vector Laboratories). Slides were counterstainedwith hemotoxylin and eosin.

Patient Studies and ELISA for ST2.

HEART Study

The Healing and Early Afterload Reducing Therapy (HEART) study was arandomized, double-blind, placebo-controlled trial that enrolled 352patients with acute myocardial infarction (MI) from 36 centers in theUnited States and Canada. Men and women over the age of 21 years who hadexperienced an MI within 24 hours were eligible. Inclusion and exclusioncriteria, and details of the trial design have been previously described(Pfeffer M. A., et al., Circulation, 1997, 95:2643-2651; Greaves S. C.,et al., Am. J. Cardiol, 1997, 80:442-448; Solomon S. D., et al., Ann.Intern. Med., 2001, 134:451-458; Aikawa Y., et al., Am. Heart J, 2001,141:234-242). Serial blood samples from days 1, 14, and 90 aftermyocardial infarction from 69 randomly chosen patients in the HEARTtrial were available for this study. Soluble T1/ST2 was assayed with adouble monoclonal sandwich ELISA assay that has been previouslydescribed (Kuroiwa K., et al., Hybridoma, 2000, 19:151-159). The assayis commercially available (MBL International, Watertown, Mass.).

PRAISE Study

The Prospective Randomized Amlodipine Survival Trial (PRAISE) study wasa prospective large-scale study of amlodipine in patients with heartfailure due to coronary artery disease. The results of this trial werenull for a benefit of Amlodipine in severe heart failure. Blood sampleswere drawn at the beginning of this study before therapy and then twicemore during the study. Soluble T1/ST2 was assayed as described above.One of the key current blood tests for heart failure is brainnatriuretic peptide (BNP). We examined whether T1/ST2 levels in heartfailure patients were altered and whether T1/ST2 levels correlated withBNP levels in these patients.

Statistics.

Each in vitro experiment shown was performed a minimum of three times.

Values are means±SEM. Data were analyzed by one-way ANOVA, or ANOVA forrepeated measures, with post hoc Bonferroni multiple comparison analyseswhere appropriate. Linear regression was performed on serum values withlog transformed values due to non-normal parameter distributions. Pvalues <0.05 were considered statistically significant.

Results:

In vivo Expression of T1/ST2 Protein in Myocardial Infarction in Mice.

To evaluate expression of T1/ST2 in injured myocardium, mice weresubjected to experimental myocardial infarction through coronary arteryligation. FIG. 11 shows protein expression of T1/ST2 usingimmunohistochemistry in mouse hearts 1 and 3 days post myocardialinfarction. Positive staining was seen 1 day post myocardial infarction(post-MI) in all regions of the left ventricle, normal, infarct andborder zones, but not at 3 days post myocardial infarction. No stainingfor T1/ST2 was observed in 1 and 3 day sham-operated controls. Theseresults suggest that T1/ST2 protein is expressed in response to acuteinjury during the early phase of post-infarction remodeling before themigration of macrophages into the infarct and border zones seen at 3days. The monoclonal antibody used for these studies does notdistinguish between soluble and membrane forms of T1/ST2.

Soluble T1/ST2 is Increased in the Systemic Circulation of Patients OneDay after Myocardial Infarction.

Since soluble T1/ST2 is highly induced in cardiac myocytes, and T1/ST2protein is highly expressed in mouse myocardium following experimentalmyocardial infarction, we hypothesized that soluble T1/ST2 is increasedin the systemic circulation of patients following myocardial infarction.

Methods and Results:

Using a double monoclonal sandwich ELISA assay, we assayed blood samplesfrom 69 participants of the HEART Study on the day of myocardialinfarction (day 1), as well as day 14 and day 90 after infarction. Asshown in FIG. 12A, systemic T1/ST2 protein was significantly increasedone day after myocardial infarction (mean±SEM, 3.8±0.4 ng/ml, p<0.001;range, 0.32 to 17.42 ng/ml) compared to day 14 (mean±SEM, 0.98±0.06ng/ml; range, 0.25 to 3.42 ng/ml) and day 90 (mean±SEM, 0.79±0.07 ng/ml;range, 0.02 to 3.53 ng/ml; day 14 vs. day 90, P=NS). Mean values at day90 were similar to published mean values for healthy controls (KuroiwaK., et al., Hybridoma, 2000, 19:151-159). Systemic T1/ST2 protein levelscorrelated positively with peak creatine kinase levels (r=0.41,p<0.001), shown in FIG. 12B. High systemic ST2 protein levels were alsoassociated with low ejection fraction one day after myocardialinfarction as shown in quartile analysis (p=0.03) in FIG. 12C and usingregression analysis in FIG. 12D.

Conclusions:

These results suggest a coordinated regulation between the extent ofmyocardial injury and synthesis and secretion of soluble T1/ST2 into thesystemic circulation in the clinical setting of myocardial infarction.

Soluble T1/ST2 is Increased in the Systemic Circulation of Patients withSevere Chronic Heart Failure.

This study tested the hypothesis that soluble T1/ST2 levels in the serumof patients with severe chronic heart failure are associated with levelsof BNP, ProANP and norepinephrine, neurohormones that are increased inheart failure.

Methods and Results:

Serum samples, clinical variables and neurohormone levels from theneurohormone substudy of the Prospective Randomized Amlodipine SurvivalEvaluation 2 study (PRAISE-2) heart failure trial (New York HeartAssociation functional class III or IV, end point: mortality ortransplantation) were used. The PRAISE-2 study was a multi-center,randomized, double blinded, parallel group, placebo-controlled study toevaluate the effects of amlodipine 10 mg/day on survival in patientswith congestive heart failure of a non-ischemic etiology. The trialconsisted of—patients recruited from 240 sites in the United States andCanada. The neurohormone substudy consisted of 181 patients recruitedfrom 26 centers participating in the main study. Both the main PRAISE-2and the neurohormonal substudy were approved by the institutional reviewboards of the participating institutions. Patients were eligible if theywere at least 18 years of age, had heart failure of a non-ischemicetiology, symptoms at rest or upon minimal exertion (New York HeartAssociation functional class III or IV) and a left ventricular ejectionfraction lower than 30%. All patients were on treatment with ACEinhibitors and digoxin for at least 3 months. Patients were excluded ifthey had a recent or remote history of angina.

Assays for T1/ST2, Neurohormones and Measurement of Oxidative Stress.

Blood samples were evaluated at baseline and 2 weeks (Table 1). SolubleT1/ST2 was measured with a sandwich double monoclonal antibody ELISAmethod (Medical & Biological Laboratories Co., Ltd., Nagoya, Japan)according to the manufacturer's instruction. In brief, serum samples orstandards were incubated in the microwells coated with anti-human T1/ST2antibody. After washing, peroxidase-conjugated anti-human T1/ST2antibody was added into the microwell and incubated. After anotherwashing, the peroxidase substrate was added and the optical density at450 nm was determined. Circulating catecholamines (norepinephrine,epinephrine, dopamine), angiotensin II, natriuretic peptides (pro-atrialnatriuretic peptide (Pro-ANP), brain natriuretic peptide (BNP)) andindices of oxidative stress (malondialdehyde, adrenolutin) were measuredas previously described (Dhalla K S, et al., Mol Cell Biochem, 1989;87:85-92; Moe G W, et al., Am Heart J, 2000; 139:587-95). T1/ST2 serummeasurements were performed on samples from 162 patients obtained attrial enrollment and from 135 of the same patients obtained 2 weeksafter trial enrollment. Baseline T1/ST2 levels correlated with baselineBNP levels (r=0.3511, p<0.0001), baseline ProANP levels (r=0.3598,p<0.0001) and baseline norepinephrine levels (r=0.3854, p<0.0001) (Table2). The change in T1/ST2 (T1/ST2 levels at 2 weeks minus T1/ST2 levelsat trial enrollment) was significant as a univariate predictor ofmortality or transplantation (p=0.048) as was baseline BNP (p<0.0001)and baseline ProANP (p<0.0001) (Table 3). In multivariate modelsincluding BNP and ProANP, the change in T1/ST2 remained significant asan independent predictor of mortality or transplantation independent ofBNP and ProANP (Table 4).

TABLE 1 Baseline Characteristics 95^(th) N Median 5^(th) PercentilePercentile A. All Patients Baseline ST2 (ng/mL) 161 0.24 0.16 0.70Baseline BNP (pmol/L) 162 56.0 3.70 264.30 Baseline ProANP (pg/L) 1621778.50 531.00 5615.00 Norepinephrine (pg/mL) 158 401.58 165.90 1096.00Dopamine (pg/mL) 158 39.06 4.22 398.40 Epinephrine (pg/mL) 158 54.9211.64 139.90 Angiotensin II (pg/mL) 157 22.60 7.00 67.30 Adrenolutin(ng/mL) 156 22.84 4.31 369.31 Creatinine (mmol/L) 158 1.10 0.80 1.90 Age(years) 157 59.9 32.5 78.2 Body Mass Index (kg/mm2) 157 27.6 20.4 39.7LV Ejection Fraction 158 22.0 11.0 30.0 B. Patients With Blood Samplesat Baseline and Week 2 Baseline ST2 (ng/mL) 135 0.24 0.15 0.81 BaselineBNP (pmol/L) 135 54.90 3.30 264.30 Baseline ProANP (pg/L) 135 1788.00488.00 4788.00 Norepinephrine (pg/mL) 130 395.05 171.70 1118.00 Dopamine(pg/mL) 130 64.02 4.32 405.50 Epinephrine (pg/mL) 130 56.07 12.24 134.80Angiotensin II (pg/mL) 131 21.70 7.00 58.30 Adrenolutin (ng/mL) 13024.41 4.43 369.31 Creatinine (mmol/L) 135 1.10 0.80 2.00 Age (years) 13460.5 34.4 78.2 Body Mass Index (kg/mm2) 134 27.4 20.5 39.7 LV EjectionFraction 135 22.0 11.0 30.0

TABLE 2 Relation of ST2 to Clinical Variables and Neurohormones:Spearman Correlations Baseline ST2 Change in ST2 Baseline BNP (pmol/L) R0.3511 −0.11327 p value <0.0001 0.1843 N 161 139 Baseline ProANP(pmol/L) R 0.35979 −0.10967 p value <0.0001 0.1987 N 161 139 Change inBNP* (pmol/L) R −0.10184 0.21497 p value 0.2329 0.0110 N 139 139 Changein ProANP* (pmol/L) R 0.05584 0.28847 p value 0.5138 0.0006 N 139 139Norepinephrine (pg/ml) R 0.38535 −0.25253 p value <0.0001 0.0032 N 156134 Dopamine (pg/mL) R 0.07879 0.22127 p value 0.3283 0.0102 N 156 134Epinephrine (pg/mL) R 0.08043 −0.12110 p value 0.3182 0.1634 N 156 134Angiotensin II (pg/mL) R 0.00374 −0.00725 p value 0.9630 0.9335 N 156135 Adrenolutin (ng/mL) R 0.00544 −0.10422 p value 0.9464 0.2308 N 155134 Creatinine (units) R 0.16567 0.02513 p value 0.0388 0.7724 N 156 135LV Ejection Fraction R −0.08006 0.03651 p value 0.3205 0.6742 N 156 135Age (years) R −0.11768 0.19260 p value 0.1447 0.0274 N 155 134 Body MassIndex (units) R 0.04561 −0.05410 p value 0.5731 0.5347 N 155 134 R,Spearman correlation coefficient; N, sample number. Baseline, values attrial enrollment; *Change, values at week 2 minus values at trialenrollment.

TABLE 3 Univariate Predictors of Mortality and Transplantation(Endpoint) 95% confidence Variable Odds Ratio interval p-value BaselineST2, per 0.1 ng/mL 1.114 0.961-1.300 0.1509 Baseline BNP, per 10 pmol/L1.106 1.060-1.161 <0.0001 Baseline ProANP, per 10 pg/L 1.007 1.005-1.010<0.0001 Change in ST2*, per change of 0.1 ng/mL 1.320 1.042-1.827 0.0482Change in BNP*, per change of 10 pmol/L 1.033 0.966-1.110 0.3401 Changein ProANP*, per change of 10 pg/L 1.003 0.997-1.009 0.3413Norepinephrine, per 1 pg/mL 1.001 1.000-1.002 0.0562 Dopamine, per 10pg/mL 1.029 1.006-1.059 0.0433 Epinephrine, per 1 pg/mL 0.9990.995-1.001 0.6645 Angiotensin II, per 1 pg/mL 0.997 0.977-1.017 0.7921Adrenolutin, per 10 ng/mL 0.985 0.943-1.017 0.4167 Creatinine, per 1mmol/L 2.487 0.997-6.417 0.0526 LV Ejection Fraction 0.952 0.897-1.0070.0906 Race 1.947 0.946-4.192 0.0776 Gender 1.225 0.576-2.728 0.6061 Age1.435 1.099-1.914 0.0104 Etiology 1.543 0.744-3.336 0.2543 Body MassIndex, per 1 kg/mm² 0.972 0.919-1.021 0.2876 Baseline, values at trialenrollment; *Change, values at week 2 minus values at trial enrollment.

TABLE 4 Multivariate Predictors of Mortality and Transplantation(Endpoint): Predictive Value of ST2 Variables P Baseline ST2 andBaseline BNP Baseline BNP 0.0003 Baseline Dopamine 0.0906 Baseline ST20.6368 Baseline ST2 and Baseline ProANP Baseline ProANP <0.0001 BaselineDopamine 0.0944 Baseline ST2 0.3306 Change in ST2* and Baseline BNPBaseline BNP 0.0001 Change in ST2 0.0392 Change in ST2* and BaselineProANP Baseline ProANP <0.0001 Change in ST2 0.0274 Baseline, values attrial enrollment; *Change, values at week 2 minus values at trialenrollment.

Example 3 Methods

Study Populations.

The Thrombolysis in Myocardial Infarction (TIMI) 14 trial was arandomized, open-label, dose-ranging study of combination reperfusiontherapy for patients with ST-segment elevation MI conducted betweenMarch 1997 and July 1998. Specifically, this study was an angiographictrial comparing 4 different thrombolytic combinations: abciximab alone,alteplase alone, abciximab with reduced dose of alteplase, and abciximabwith reduced dose of streptokinase (Antman E M et al., Circulation,1999; 99:2720-32; Antman E M et al., Eur Heart J, 2000; 21:1944-53). TheENTIRE-TIMI 23 trial was an open-label, dose-ranging, multicenter studyconducted between February 2000 and September 2001 to evaluateenoxaparin as adjunctive antithrombin therapy with various forms ofpharmacological reperfusion, including full-dose tenecteplase andhalf-dose tenecteplase plus abciximab (Antman E M et al., Circulation.2002; 105:1642-9). In both studies, patients were eligible for inclusionif they had a qualifying episode of ischemic discomfort of at least 30min within 6 hr (ENTIRE) or 12 hr (TIMI 14), and exhibited at least 0.1mV ST-segment elevation in 2 contiguous precordial electrocardiographicleads. Exclusion criteria for both trials included increased risk ofhemorrhage, severe renal insufficiency, and cardiogenic shock.

Laboratory Analyses.

Serum samples collected at baseline, 1, 3, 12, and 24 hr afterenrollment in TIMI 14 were evaluated. Serum samples from the ENTIREtrial were available only at baseline. Serum was isolated within 60 minof sample collection and stored at −20° C. or colder until shipped tothe TIMI Biomarker Core Lab (Boston, Mass.), where samples weremaintained at −70° C. Soluble ST2 was measured with a sandwich doublemonoclonal antibody ELISA method (Medical & Biological Laboratories Co.,Ltd., Nagoya, Japan). Serum samples or standards were incubated inmicrowells coated with anti-human ST2 antibody. After washing,peroxidase-conjugated anti-human ST2 antibody was added into themicrowell and incubated. After washing again, the peroxidase substratewas added and the optical density at 450 nm was determined. Highsensitivity C-reactive protein (hs-CRP, Dade-Behring Inc, Deerfield,Ill.), creatine kinase MB isoenzyme (CK-MB), B-type natriuretic peptide(SHIONORIA BNP, Shionogi, Osaka, Japan). and cardiac troponin I (ACS:180, Bayer Diagnostics, Tarrytown, N.Y.) were measured using previouslydescribed methods (Morrow D A et al., J Am Coll Cardiol. 1998;31:1460-5; Morrow D A et al., Clin Chem. 2000; 46:453-460). Creatinekinase isoenzyme levels were measured locally at the site on admission,at 3 hours, and at 6 to 8 hour intervals for the first 24 hours. Due tosample availability, BNP levels were measured in samples fromENTIRE-TIMI 23, but not TIMI 14.

Statistical Analysis.

Patients were divided into quartiles on the basis of their ST2 serumlevels at the time of enrollment into the studies. ST2 levels aredescribed using the median and 25^(th)-75^(th) percentiles. Theassociation between baseline clinical characteristics and quartiles ofST2 were analyzed using the Kruskal-Wallis test for continuous variablesand the χ² test for categorical variables. Correlations between ST2 andother continuous baseline variables were studied with a non-parametric(Spearman's) correlation coefficient. For evaluation of association withclinical outcomes, ST2 was compared between patients who met a study endpoint and those who did not using the Wilcoxon rank-sum test.Multivariable analysis of the association of ST2 with outcomes wasperformed using logistic regression including terms for establishedpredictors of mortality in ST-elevation myocardial infarction (STEMI)(Morrow, D A et al., Circulation 2000; October 24; 102(17):2031-7).Except where stated, results presented are for the combined TIMI 14 andENTIRE-TIMI 23 study population.

Results

Baseline ST2 and Clinical Variables.

Most baseline clinical characteristics, including gender, age, weight,and extent of coronary artery disease did not correlate with baselineST2 levels (Table 5). Few patients in this population had either a priorhistory or presented with clinical evidence of heart failure.Interestingly, heart rate correlated positively with ST2 levels(p<0.0001) and systolic blood pressure showed a modest correlation withST2 levels (p=0.05), consistent with the theory that ST2 is secreted bycardiac myocytes under biomechanical stress. The biomarkers cardiactroponin I, BNP, and CRP—which have all been shown to predict outcomeafter myocardial infarction (de Lemos J A et al., N Engl J Med 2001;345:1014-21; Antman E M et al, N Engl J Med 1996; 335:1342-9; Morrow D Aet al., J Am Coll Cardiol 1998; 31:1460-5) were correlated with ST2 byquartile analysis and cardiac troponin I and CRP were statisticallysignificant. When these biomarkers were evaluated as continuousvariables, quantitatively weak correlations were observed (Table 6).

TABLE 5 Baseline Clinical Characteristics According to Quartiles of ST2(ng/mL) Quartile 1 Quartile 2 Quartile 3 Quartile 4 p trend p Q4 vs Q1Range, ng/mL 0.085-0.179 0.180-0.235 0.236-0.346 0.347-6.88 n 204 202202 202 Time CP to 2.8±1.6 3.1 ± 1.5 3.2 ± 1.4 4.0 ± 1.9 <0.0001 <0.0001randomization (hrs) Age (years) 58 ± 10 58 ± 10 58 ± 11 58 ± 10 0.9 1.0Male 74% 77% 85% 81% 0.03 0.09 White 88% 89% 90% 88% 0.9 1.0 PastMedical History Hypertension 25% 24% 36% 33% 0.02 0.09 Congestive HeartFailure  0%  0% 1.5%  1.0%  0.1 0.2 Angina 26% 24% 26% 32% 0.3 0.2Diabetes 14% 14% 15% 16% 0.9 0.5 Family history of CAD 73% 73% 73% 73%0.2 0.08 Hypercholesterolemia 22% 21% 21% 29% 0.2 0.1 Smoking status:Current smoker 57% 48% 49% 48% 0.2 0.06 Physical findings Weight kg 83 ±16 81 ± 15 82 ± 14 83 ± 15 0.4 0.8 Systolic BP (mm Hg) 139 ± 21  138 ±22  141 ± 23  143 ± 22  0.1 0.05 HR (BPM) 71 ± 17 75 ± 17 72 ± 16 80 ±17 0.001 <0.0001 Killip Class II-IV 2.0%  1.5%  3.6%  4.5%  0.3 0.2Diagnostic Testing cTnI > 0.1 ng/ml* 61% 69% 77% 84% 0.001 <0.0001 BNP >80 pg/ml* 1.8%  5.4%  7.2%  14.4%  0.003 0.001 CRP > 1.5 ng/ml 2.1% 8.8%  8.1%  11.4%  0.006 <0.0001 Creatinine mg/dL  1.0 ± 0.21  1.0 ±0.20  1.0 ± 0.25  1.1 ± 0.28 0.1 0.03 Extent CAD (50% 0.3 0.2 stenosis)1 vessel 48% 55% 45% 50% 2 vessel 38% 28% 34% 30% 3 vessel 15% 18% 20%20% EF (%)** 58 ± 15 58 ± 15 57 ± 15 57 ± 15 1.0 0.9 CP = Chest Pain; HR= Heart Rate; cTnI = Cardiac Troponin I; BNP = B type NatriureticPeptide; CRP = C Reactive Protein; CAD = Coronary Artery Disease; EF =Ejection Fraction *Measured in the ENTIRE-TIMI 23 population only; N =448 except **(N = 469)

TABLE 6 Correlation between ST2 and Continuous Variables VariableSpearmans rho P value Time CP to randomization 0.29 <0.0001 Age −0.0030.9 Weight (kg) 0.01 0.8 CKMB peak 0.08 0.02 cTnI* 0.26 <0.0001 CRP 0.100.007 BNP* 0.068 0.15 Creatinine 0.09 0.01 LVEF** −0.005 0.9 CP = ChestPain; CKMB = MB isoenzyme of creatine kinase; cTnI = Cardiac Troponin I;BNP = B type Natriuretic Peptide; CRP = C Reactive Protein; CAD =Coronary Artery Disease; EF = Ejection Fraction. *Measured in theENTIRE-TIMI 23 population only; N = 448 except **(N = 469)

ST2 and Clinical Outcomes.

For the combined cohort of 810 patients, baseline ST2 was significantlyassociated with clinical outcomes at 30 days (Table 7). Specifically,levels of ST2 were significantly higher at presentation among patientswho subsequently died (p=0.0001), or developed new or worsening CHF(p=0.009), by 30 days after enrollment. Dichotomized at the median,elevated baseline levels of ST2 were indicative of higher mortalitythrough 30 days of follow-up (log-rank, p=0.0009, FIG. 13). Moreover, inan analysis by quartiles of ST2, the risk of both death (p=0.001) andthe composite of death or CHF (p=0.001) increased in a graded, stepwisefashion with higher levels of ST2. This association between ST2 andclinical events was homogeneous between the two individual trials (TIMI14 and ENTIRE-TIMI 23).

TABLE 7 Association between Baseline ST-2 Concentration (ng/ml) andOutcomes Outcome (30 days) n Median [25, 75] p value Dead 28 0.379[0.267, 0.611] 0.0001 Alive 782 0.233 [0.178, 0.340] MI 29 0.213 [0.171,0.259] 0.11 No MI 781 0.237 [0.181, 0.348] CHF 21 0.287 [0.237, 0.470]0.009 No CHF 789 0.233 [0.178, 0.345] Death/CHF 47 0.317 [0.246, 0.590]<0.0001 No Death/CHF 763 0.231 [0.177, 0.339] MI = MyocardialInfarction; CHF = Congestive Heart Failure

Evolution of ST2 Serum Levels.

Baseline ST2 levels analyzed by quartile were significantly correlatedwith the time to randomization (Tables 5 and 6). ST2 levels wereanticipated to increase in the first day following coronary occlusionand return to normal over the next 14 days (6). Among the TIMI 14patients, analysis of serial measurements of serum ST2 in 228 patientsrevealed an increase with time, with most patients reaching a peak ST2level at 12 hours, although, a few patients had ST2 serum levels thatcontinued to increase past this time point.

Multivariate Analysis.

After controlling for established clinical predictors in STEMI includingage, heart rate, systolic blood pressure, location of myocardialinfarction, Killip class, and time from onset of chest pain, increasinglevels of ST2 remained an independent predictor of death at 30 days (OR1.77; 95% CI 1.01-3.12, p=0.047). This association was no longersignificant when BNP was added to the clinical model (assessment waslimited to ENTIRE). The predictive capacity of ST2 ascertained at latertime points (3 and 12 hours in TIMI 14) was also evaluated; revealing astronger association between ST2 and mortality risk.

Serum soluble T1/ST2, therefore is a novel biomarker for severe heartfailure that parallels neurohormonal activation. In patients with severechronic NYHA Class III-IV heart failure, the change in T1/ST2 levels isan independent predictor of the endpoint of mortality ortransplantation.

In this study, we explored the potential role of serum measurement of arecently-identified receptor of the interleukin-1 family in acutemyocardial infarction. The soluble form of this receptor is rapidlysecreted by cardiac myocytes when the cells are biomechanicallyoverloaded; this suggests that the receptor may play a role inconditions where the myocardium is rapidly overloaded, such as inmyocardial infarction. To explore this, we measured serum ST2 levels atthe time of presentation in a cohort of patients with acute myocardialinfarction. The results demonstrate that ST2 levels at the time ofpresentation in these patients are associated with in-hospital and30-day mortality. Furthermore, multivariate analysis indicated that ST2level is independently associated with outcome after controlling forimportant clinical factors.

Thus, the significance of these data is twofold. Foremost, these datasuggest that the interleukin receptor family, which participates in hostdefense and differentiation of T cells (Sims J E. IL-1 and IL-18receptors, and their extended family. Curr Opin Immunol. 2002;14:117-22), may participate in early events in acute myocardialinfarction. These data implicate this receptor as a potential noveltarget for modifying prognosis in patients with myocardial infarction.Secondarily, ST2 represents a novel biomarker that offers prognosticinformation in patients with acute myocardial infarction; thus,extending upon our prior work demonstrating an association between ST2and mortality among patients with non-ischemic congestive heart failure(Weinberg E O, Shimpo M, Hurwitz S, Tominaga S, Rouleau J L, Lee R T.Identification of serum soluble ST2 receptor as a novel heart failurebiomarker. Circulation. 2003; 107:721-6), another condition ofmyocardial overload.

Although not excluded, it is unlikely that the relationship of ST2 andoutcome after myocardial infarction is simply a reflection of theassociation of chronic elevations in inflammatory markers like CRP andrisk of myocardial infarction. ST2, like BNP, may be synthesized bycardiac myocytes themselves and data from patients without apparentischemic disease suggests that ST2 predicts prognosis in the absence ofcoronary artery disease. Furthermore, preliminary data suggest that ST2levels in outpatients with stable coronary artery disease are unrelatedto CRP levels. While our data support the complementary value of ST2 forrisk assessment when added to a robust clinical model (REF TIMI RiskScore), ST2 did not contribute additional information to BNP in thesmaller data set limited to ENTIRE-TIMI 23. There may also be prognosticvalue of ST2 in conjunction with other available biomarkers.

Although ST2 may be secreted by mechanically-overloaded cardiacmyocytes, many cells can secrete ST2. It is therefore possible thatelevations in serum ST2 are not completely specific for acute myocardialinfarction. In addition to non-ischemic heart failure (Weinberg E O,Shimpo M, Hurwitz S, Tominaga S, Rouleau J L, Lee R T. Identification ofserum soluble ST2 receptor as a novel heart failure biomarker.Circulation. 2003; 107:721-6), patients with asthma (Oshikawa K, KuroiwaK, Tago K, Iwahana H, Yanagisawa K, Ohno S, Tominaga S I, Sugiyama Y.Elevated soluble ST2 protein levels in sera of patients with asthma withan acute exacerbation. Am J Respir Crit Care Med. 2001; 164:277-81) orautoimmune diseases like systemic lupus erythematosus (Kuroiwa K, AraiT, Okazaki H, Minota S, Tominaga S. Identification of human ST2 proteinin the sera of patients with autoimmune diseases. Biochem Biophys ResCommun. 2001; 284:1104-8) may also have increased serum ST2 levels.Therefore, the usefulness of ST2 measurement in the initial diagnosis ofacute myocardial infarction in such subjects is not unequivocal.

However, ST2 remains a possible target for therapy in patients with MI.These data demonstrate how genomic technology can reveal a new potentialpathophysiological pathway in a common disease. ST2 was initiallyidentified through studies of the interleukin-1 family, but its role inmyocardial disease was only recently suggested by genomic studies withDNA microarrays. Studies with DNA microarrays allow identification ofpotential new disease pathways, but this is only an initial step inunderstanding the role of the pathway. The above data supports the rolefor ST2 in acute myocardial infarction, since the levels of ST2 predictoutcome. Studies of the function of ST2 in myocardial infarction arepossible. In addition, identifying the ligand for the soluble andmembrane ST2 receptors could help further the understanding of thepotentially competing roles of the membrane and soluble receptors.

The results described establish that the T1/ST2 is secreted during aheart attack and/or heart failure, and can be easily measured, therebysupporting the asserted utilities of the invention.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

All references disclosed herein are incorporated by reference in theirentirety.

1.-33. (canceled)
 34. A method for selecting a beta-adrenergic receptorblocker or an angiotensin system inhibitor for a subject, and treatingthe subject, the method comprising: (a) performing an assay to determinea level of soluble ST2 in a biological sample from a subject; (b)identifying a subject that has an elevated level of soluble ST2 ascompared to a predetermined value of soluble ST2; (c) selecting abeta-adrenergic receptor blocker of an angiotensin system inhibitor forthe identified subject; and (d) administering a therapeuticallyeffective amount of the selected beta-adrenergic receptor blocker orangiotensin system inhibitor to the identified subject.
 35. The methodof claim 34, wherein the biological sample is blood or serum.
 36. Themethod of claim 34, wherein the assay is performed using an antibody oran antibody fragment that specifically binds to soluble ST2.
 37. Themethod of claim 34, wherein the predetermined value of soluble ST2 is alevel of soluble ST2 in a healthy subject.
 38. The method of claim 34,wherein the subject is human.
 39. The method of claim 38, wherein thesubject is hypercholesterolemic, hypertriglyceridemic, hyperlipidemic, asmoker, or hypertensive.
 40. The method of claim 34, wherein the subjectis free of symptoms calling for treatment for a cardiovascular disorder.41. The method of claim 34, wherein step (d) comprises administering theselected beta-adrenergic receptor blocker to the identified subject. 42.The method of claim 41, wherein the beta-adrenergic receptor blocker isselected from the group consisting of: atenolol, acebutolol, alprenolol,befunolol, betanxolol, bunitrolol, carteolol, celiprolol, hedroxalol,indenolol, labetalol, levobunolol, mepindolol, methylpranol, metindol,metoprolol, metrizoranolol, oxpresnolol, pindolol, propranolol,practolol, sotalonadolol, tiprenolol, tomalolol, timolol, bupranolol,penbutolol, trimepranol,2-(3-(1,1-dimethyl-ethyl)-amino-2-hydroxypropoxy)-3-pyridenecarbonitril,1-butylamino-3-(2,5-dichlorophenoxy)-2-propanol,1-iso-propylamino-3-(4-(2-cyclopropylmethoxyethyl) phenoxy)-2-propanol,3-isopropylamino-1-(7-methyllindan-4-yloxy)-2-butanol,2-(3-t-butylamino-2-hydroxy-propylthio)-4-(5-carbamoyl-2-thienyl)thiazol,and 7-(2-hydroxy-3-t-butyladminopropoxy)phthalide.
 43. The method ofclaim 34, wherein step (d) comprises administering the selectedangiotensin system inhibitor to the identified subject.
 44. The methodof claim 43, wherein the angiotensin system inhibitor is selected fromthe group consisting of: angiotensin-converting enzyme (ACE) inhibitors,angiotensin II antagonists, angiotensin II receptor antagonists, agentsthat activate the catabolism of angiotensin II, and agents that preventthe synthesis of angiotensin I.
 45. A method of selecting a subject fortreatment with a beta-adrenergic receptor blocker or an angiotensinsystem inhibitor and treating the subject, the method comprising: (a)performing an assay to determine a level of soluble ST2 in a biologicalsample from a subject; (b) identifying a subject that has an elevatedlevel of soluble ST2 as compared to a predetermined value of solubleST2; (c) selecting the identified subject for treatment with abeta-adrenergic receptor blocker of an angiotensin system inhibitor; and(d) administering a therapeutically effective amount of thebeta-adrenergic receptor blocker or angiotensin system inhibitor to theselected subject.
 46. The method of claim 45, wherein the biologicalsample is blood or serum.
 47. The method of claim 45, wherein the assayis performed using an antibody or an antibody fragment that specificallybinds to soluble ST2.
 48. The method of claim 45, wherein thepredetermined value of soluble ST2 is a level of soluble ST2 in ahealthy subject.
 49. The method of claim 45, wherein the subject ishuman.
 50. The method of claim 49, wherein the subject ishypercholesterolemic, hypertriglyceridemic, hyperlipidemic, a smoker, orhypertensive.
 51. The method of claim 45, wherein the subject is free ofsymptoms calling for treatment for a cardiovascular disorder.
 52. Themethod of claim 45, wherein step (d) comprises administering thebeta-adrenergic receptor blocker to the selected subject.
 53. The methodof claim 52, wherein the beta-adrenergic receptor blocker is selectedfrom the group consisting of: atenolol, acebutolol, alprenolol,befunolol, betanxolol, bunitrolol, carteolol, celiprolol, hedroxalol,indenolol, labetalol, levobunolol, mepindolol, methylpranol, metindol,metoprolol, metrizoranolol, oxpresnolol, pindolol, propranolol,practolol, sotalonadolol, tiprenolol, tomalolol, timolol, bupranolol,penbutolol, trimepranol,2-(3-(1,1-dimethyl-ethyl)-amino-2-hydroxypropoxy)-3-pyridenecarbonitril,1-butylamino-3-(2,5-dichlorophenoxy)-2-propanol,1-iso-propylamino-3-(4-(2-cyclopropylmethoxyethyl) phenoxy)-2-propanol,3-isopropylamino-1-(7-methyllindan-4-yloxy)-2-butanol,2-(3-t-butylamino-2-hydroxy-propylthio)-4-(5-carbamoyl-2-thienyl)thiazol,and 7-(2-hydroxy-3-t-butyladminopropoxy)phthalide.
 54. The method ofclaim 45, wherein step (d) comprises administering the angiotensinsystem inhibitor to the selected subject.
 55. The method of claim 54,wherein the angiotensin system inhibitor is selected from the groupconsisting of: angiotensin-converting enzyme (ACE) inhibitors,angiotensin II antagonists, angiotensin II receptor antagonists, agentsthat activate the catabolism of angiotensin II, and agents that preventthe synthesis of angiotensin I.